Isoprene oligomer, polyisoprene, processes for producing these materials, rubber composition, and pneumatic tire

ABSTRACT

The invention relates to an isoprene oligomer that contains a trans structural moiety and a cis structural moiety, which can be represented by the following formula (1), wherein at least 1 atom or group in the trans structural moiety is replaced by another atom or group. The invention also relates to a polyisoprene, which is biosynthesized using the isoprene oligomer and isopentenyl diphosphate. Further, this invention provides a rubber composition comprising the isoprene oligomer and/or the polyisoprene, and a pneumatic tire, including tire components (e.g., treads and sidewalls) formed from the rubber composition. 
                         
wherein n represents an integer from 1 to 10; m represents an integer from 1 to 30; and Y represents a hydroxy group, a formyl group, a carboxy group, an ester group, a carbonyl group, or a group represented by the following formula (2):

This application is a Divisional of copending application Ser. No.13/809,616, filed on Jan. 11, 2013, which was filed as PCT InternationalApplication No. PCT/JP2011/064774 on Jun. 28, 2011, which claims thebenefit under 35 U.S.C. §119(a) to Patent Application No. 2010-160120,filed in Japan on Jul. 14, 2010 and Patent Application No. 2010-277384,filed in Japan on Dec. 13, 2010, all of which are hereby expresslyincorporated by reference into the present application.

TECHNICAL FIELD

The present invention relates to an isoprene oligomer and apolyisoprene; processes for producing these materials; a rubbercomposition including the isoprene oligomer and/or the polyisoprene; anda pneumatic tire formed from the rubber composition.

BACKGROUND ART

Conventionally, in order to impart novel properties, in addition to theoriginal properties of rubber, to rubber products, filler and the likeof various materials or shapes are introduced into rubber compositionsaccording to the uses to achieve the desired properties. For automobiletires, for example, in order to improve properties such as abrasionresistance, low-heat-build-up properties, or wet grip performance,filler (e.g., silica, carbon black) is introduced into the organicrubber fraction.

When filler or the like is mixed with the rubber fraction in such arubber composition, for the purpose of enhancing the affinity betweenthe two to improve low-heat-build-up properties, wet grip performance,etc., a modified rubber (modified diene polymer) has been used in whicha functional group having affinity for the filler is introduced inrubber molecules in the rubber fraction as a result of, for example, atreatment involving reacting rubber molecules with, for example, acompound containing both a nitrogen atom-containing group and achlorosulfenyl group (see, for example, Patent Literatures 1 and 2).

It is however known that, depending on methods for introduction, apredetermined functional group is difficult to introduce to apredetermined position in the rubber molecule and is consequentlyintroduced to, particularly, a random position in the main chainconstituting the rubber molecule. Use of such rubber moleculescontaining a predetermined functional group introduced in the main chainis less likely to produce the desired effects due to the random bondsbetween the rubber molecules and filler. In addition, the functionalgroup-introduced sites have deteriorated rubber properties, whichdisadvantageously results in deterioration in the properties of thewhole rubber.

CITATION LIST Patent Literature

-   Patent Literature 1: JP 2000-001573 A-   Patent Literature 2: JP 2000-001575 A

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to solve the problems describedabove and to provide an isoprene oligomer and a polyisoprene which havebeen modified substantially only at a terminal site of the molecule.Another object of the present invention is to provide a rubbercomposition including the isoprene oligomer and/or the polyisoprene, andpneumatic tires including various tire components (e.g., treads andsidewalls) formed from the rubber composition.

Solution to Problem

The present invention relates to an isoprene oligomer including a transstructural moiety and a cis structural moiety, which is represented bythe following formula (1), wherein at least 1 atom or group in the transstructural moiety is replaced by another atom or group:

wherein n represents an integer from 1 to 10; m represents an integerfrom 1 to 30; and Y represents a hydroxy group, a formyl group, acarboxy group, an ester group, a carbonyl group, or a group representedby the following formula (2):

It is preferable that at least 1 atom or group in moiety II in thefollowing formula (1-1) should be replaced, and no atom or group inmoiety III should be replaced:

It is preferable that the trans structural moiety should be representedby any of the following formulas (a) to (s):

It is preferable that the isoprene oligomer should be biosynthesizedusing an allylic diphosphate and isopentenyl diphosphate; the allylicdiphosphate is represented by the following formula (3), wherein atleast 1 atom or group in the isoprene units in formula (3) is replacedby another atom or group:

wherein p represents an integer from 1 to 10.

It is preferable that the biosynthesis should be carried out using anenzyme that shows prenyltransferase activity.

It is preferable that the enzyme that shows prenyltransferase activityshould be any of the following proteins:

[1] a protein having an amino acid sequence represented by any of thefollowing SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22;

[2] a protein having an amino sequence that is derived from the aminoacid sequence represented by any of the following SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, and 22 by substitution, deletion, insertion,or addition of 1 or more amino acids, and possessing the ability tocatalyze a reaction between an allylic diphosphate and isopentenyldiphosphate, wherein the allylic diphosphate can be represented by thefollowing formula (3), wherein at least 1 atom or group in the isopreneunits in formula (3) is replaced by another atom or group;[3] a protein having an amino acid sequence that shows 45% or highersequence identity to the amino acid sequence represented by any of thefollowing SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 andpossessing the ability to catalyze a reaction between an allylicdiphosphate and isopentenyl diphosphate, wherein the allylic diphosphatecan be represented by the following formula (3), wherein at least 1 atomor group in the isoprene units in formula (3) is replaced by anotheratom or group:

wherein p represents an integer from 1 to 10.

The present invention also relates to a process for producing theisoprene oligomer, which involves the biosynthesis of isoprene oligomerby using an allylic diphosphate and isopentenyl diphosphate, wherein theallylic diphosphate can be represented by the following formula (3),wherein at least 1 atom or group in the isoprene units in formula (3) isreplaced by another atom or group:

wherein p represents an integer from 1 to 10.

It is preferable that the biosynthesis should be carried out using anenzyme showing prenyltransferase activity.

The present invention further relates to a polyisoprene including atrans structural moiety and a cis structural moiety, which isrepresented by the following formula (4), wherein at least 1 atom orgroup in the trans structural moiety is replaced by another atom orgroup:

wherein n represents an integer from 1 to 10; q represents an integerfrom 30 to 40 000; and Y represents a hydroxy group, a formyl group, acarboxy group, an ester group, a carbonyl group, or a group representedby the following formula (2):

It is preferable that at least 1 atom or group in moiety VI in thefollowing formula (4-1) should be replaced, and no atom or group inmoiety VII should be replaced:

It is preferable that the polyisoprene should be biosynthesized usingthe isoprene oligomer and isopentenyl diphosphate.

The present invention further relates to a process for producing thepolyisoprene, which involves the biosynthesis of the polyisoprene byusing the isoprene oligomer and isopentenyl diphosphate.

The present invention further relates to a rubber composition includingat least 1 of the isoprene oligomers and the polyisoprene.

The present invention further relates to a pneumatic tire formed fromthe rubber composition.

Advantageous Effects of Invention

The isoprene oligomer of the present invention is an isoprene oligomercontaining a trans structural moiety and a cis structural moietyrepresented by the formula (1), wherein at least one atom or groupcontained in the trans structural moiety is replaced by another atom orgroup. Moreover, the polyisoprene of the present invention is apolyisoprene containing a trans structural moiety and a cis structuralmoiety represented by the formula (4), wherein at least one atom orgroup contained in the trans structural moiety is replaced by anotheratom or group. Thus, the isoprene oligomer of the present invention andthe polyisoprene of the present invention have been modifiedsubstantially only at a terminal site of the molecule (rubber molecule)and therefore are excellent in affinity for filler such as silica whilethe original properties of the molecules (rubber molecules) are notimpaired. Accordingly, a rubber composition containing the isopreneoligomer of the present invention and/or the polyisoprene of the presentinvention is obtained as a rubber composition in which rubber moleculesare combined with filler in a level higher than ever. Thus, the presentinvention can provide a rubber composition excellent in, for example,low-heat-build-up properties, and wet grip performance. Use of therubber composition in various tire components (e.g., treads andsidewalls) can provide pneumatic tires excellent in, for example,low-heat-build-up properties, and wet grip performance.

DESCRIPTION OF EMBODIMENTS

In the process of artificially biosynthesizing a rubber molecule(polyisoprene), an enzyme such as prenyltransferase is allowed to act ona mixture of an initiating substrate such as farnesyl diphosphate (FPP)and monomer such as isopentenyl diphosphate to produce an isopreneoligomer having approximately 8 isoprene units addition-polymerized tothe initiating substrate. It is known that the isoprene oligomer is thenfurther mixed with a latex component containing an enzyme foraddition-polymerizing isopentenyl diphosphate to produce a polyisoprenehaving a large number of isopentenyl diphosphate units linked to theoligomer.

As described above, the addition polymerization mediated by naturalenzymes is indispensable to various steps for sequentially linking themonomers starting from the initiating substrate to form a rubbermolecule.

For this reason, the biosynthesis of a rubber molecule (polyisoprene)requires using an initiating substrate and monomer whose reaction can becatalyzed by the enzyme used, which results in limitations to thestructures of the initiating substrate and monomer used as raw materialsfor the rubber molecule (polyisoprene). Particularly, the initiatingsubstrate is limited to naturally occurring dimethylallyl diphosphate,geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate,and the like due to restrictions attributed to the enzyme for producingthe oligomer.

As a result, the artificially biosynthesized rubber molecule(polyisoprene) is also limited in the degree of freedom of itsstructure. Thus, free molecular design for imparting functionality thatis absent in natural rubber is made difficult.

Conventionally, in order to obtain, for example, a rubber molecule(polyisoprene) in which a functional group or the like is introduced, afunctional group having affinity for filler is introduced into a rubbermolecule, for example, by a treatment including reacting a rubbermolecule (polyisoprene) biosynthesized in advance with, for example, acompound containing both a nitrogen atom-containing group and achlorosulfenyl group, similarly as in the case of using synthetic rubberas a raw material.

In contrast, the present invention is based on the findings that anisoprene oligomer or polyisoprene having a terminal site to whichfunctionality has been imparted can be produced by using partiallystructurally modified farnesyl diphosphate or the like as an initiatingsubstrate for the isoprene oligomer or polyisoprene production.

In particular, the present invention is based on the findings that bymaintaining the structure of moiety I in the following formula (I) inthe naturally occurring initiating substrate farnesyl diphosphate or thelike, even when a desired structure is introduced in a moiety other thanmoiety I, it is possible to produce isoprene oligomers in the presenceof the naturally occurring oligomer-producing enzyme prenyltransferaseor any enzyme obtained by partial mutation thereof. This is probably,albeit not absolutely clear, because prenyltransferase is adsorbed onthe structure of moiety I in the formula (I) of the initiating substrateand is relatively insensitive to the structures of other moieties.

On the basis of these findings, an isoprene oligomer or polyisoprenehaving a terminal site having desired properties can be provided. Inother words, isoprene oligomers or polyisoprenes having variousfunctions can be provided without impairing the properties of isopreneoligomer or polyisoprene itself.

(Isoprene Oligomer)

The isoprene oligomer of the present invention is an isoprene oligomercontaining a trans structural moiety and a cis structural moietyrepresented by the formula (1) shown below, wherein at least one atom orgroup contained in the trans structural moiety is replaced by anotheratom or group. In this context, the trans structural moiety means amoiety of repeated isoprene units with a trans structure (moiety A inthe formula (1) shown below). Also, the cis structural moiety means amoiety of repeated isoprene units with a cis structure (moiety ( )_(m)(moiety B) in the formula (1) shown below). In the presentspecification, the group represented by the formula (2) shown below hasthree hydroxy groups bonded to a phosphorus atom, and some (or one) orall of these hydroxy groups are dissociated in an aqueous solution (forexample, a group represented by the formula (5) shown below is thenformed). In the present specification, the group represented by theformula (2) also conceptually encompasses such groups in which some (orone) or all of the hydroxy groups have been dissociated.

In the present specification, the modification of a terminal site of themolecule (rubber molecule) refers to the introduction of a desiredfunctional group into a predetermined site in the trans structuralmoiety present at an end of the molecule (rubber molecule), or theintroduction of a different structure into a predetermined site in thetrans structural moiety present at an end of the molecule (rubbermolecule).

wherein n represents an integer of 1 to 10; m represents an integer of 1to 30; and Y represents a hydroxy group, a formyl group, a carboxygroup, an ester group, a carbonyl group, or a group represented by thefollowing formula (2).

The isoprene oligomer of the present invention is structurally similarto natural rubber and is highly compatible with rubber molecules.Moreover, the isoprene oligomer of the present invention has beenmodified substantially only at a terminal site of the molecule.Specifically, the isoprene oligomer of the present invention contains ahydroxy group, a formyl group, a carboxy group, an ester group, acarbonyl group, or a group represented by the formula (2), positioned atthe end of the cis structural moiety, and at least one atom or groupcontained in the trans structural moiety is replaced by another atom orgroup. Thus, the isoprene oligomer of the present invention stronglyinteracts with filler such as silica while the original properties ofisoprene oligomer are not impaired. Since the isoprene oligomer of thepresent invention is highly compatible with rubber and stronglyinteracts with filler such as silica, as described above, a rubbercomposition containing this isoprene oligomer is obtained as a rubbercomposition in which rubber molecules are combined with filler in alevel higher than ever. The resulting rubber composition can have, forexample, improved low-heat-build-up properties, wet grip performance,and abrasion resistance.

The isoprene oligomer of the present invention contains a polar group orthe like only at the terminal site of the cis structural moiety and asite close to the end of the trans structural moiety. The isopreneoligomer therefore offers high dispersibility of filler such as silicaand has a large effect of improving, for example, low-heat-build-upproperties, wet grip performance, and abrasion resistance while theoriginal properties of isoprene oligomer are not impaired, compared withisoprene oligomers containing a polar group or the like in the mainchain moiety or only at the terminal site of the cis structural moiety.

Furthermore, the isoprene oligomer of the present invention hasexcellent antimicrobial activity. This is presumably because theisoprene oligomer of the present invention, in which at least one atomor group contained in the trans structural moiety is replaced by anotheratom or group, structurally differs from usual isoprene oligomerspresent in the natural world and thus has effects such as the inhibitionof enzymes or coenzymes, the inhibition of nucleic acid synthesis, theinhibition of cell membrane synthesis, the inhibition of cytoplasmicmembrane synthesis, cell membrane disruption, and cytoplasmic membranedisruption in microbes.

In the formula (1), n represents an integer of 1 to 10 (preferably 1 to4, more preferably 1 to 3).

In the formula (1), m represents an integer of 1 to 30 (preferably 1 to10, more preferably 1 to 8).

In the formula (1), Y represents a hydroxy group (—OH), a formyl group(—CHO), a carboxy group (—COOH), an ester group (—COOR), a carbonylgroup (—COR), or a group represented by the formula (2).

In the ester group (—COOR) or the carbonyl group (—COR), R represents analkyl group having 1 to 30 (preferably 1 to 17) carbon atoms. Examplesof the alkyl group having 1 to 30 carbon atoms include methyl, ethyl,propyl, butyl, and pentyl groups.

In the formula (1), Y is preferably a hydroxy group or a carboxy group,because the resulting isoprene oligomer has excellent antimicrobialproperties and strongly interacts with filler such as silica.

At least one atom or group contained in the trans structural moiety inthe formula (1) has been replaced by another atom or group.

Examples of the atom or group (atom or group before replacement)contained in the trans structural moiety include a hydrogen atom, amethyl group, a methylene group, a carbon atom, and a methine group.

Examples of the another atom include nitrogen, oxygen, sulfur, silicon,and carbon atoms. Among them, a nitrogen atom has a strongintermolecular force and causes strong interaction with enzymes or cellmembranes. For this reason, a nitrogen atom is preferred in terms ofantimicrobial properties.

Examples of the another group include an acetoxy group, alkoxy groups(preferably, alkoxy groups having 1 to 3 carbon atoms, more preferably amethoxy group), a hydroxy group, aryl groups (preferably, a phenylgroup), alkyl groups (preferably, alkyl groups having 1 to 5 carbonatoms, more preferably an ethyl group and a tert-butyl group), an acetylgroup, N-alkyl-acetamino groups (whose alkyl preferably has 1 to 5carbon atoms), and an azide group.

Particularly, since a nitrogen atom has a strong intermolecular forceand causes strong interaction with enzymes or cell membranes,N-alkyl-acetamino groups (more preferably, N-methyl-acetamino andN-butyl-acetamino groups) and azide group are preferred in terms ofantimicrobial properties.

As described above, at least one atom or group contained in the transstructural moiety has been replaced by another atom or group. For thisreplacement, it is preferable that in the moiety of repeated isopreneunits in the trans structural moiety, at least one atom or groupcontained in moiety II in the formula (1-1) shown below should bereplaced while no atom or group contained in moiety III in the formula(1-1) should be replaced. This is based on the findings by the presentinventors that by maintaining the structure of moiety I in the formula(I) in the naturally occurring initiating substrate farnesyl diphosphateor the like, even when a desired structure is introduced in a moietyother than moiety I, it is possible to produce isoprene oligomers in thepresence of the naturally occurring oligomer-producing enzymeprenyltransferase or any enzyme obtained by partial mutation thereof.

wherein n, m, and Y are as defined for n, m, and Y in the formula (1).

Specific examples of the trans structural moiety in the formula (1)include structures represented by the formulas (a) to (s) shown below.Among them, structures represented by the formulas (c), (d), (e), (f),(k), (l), and (r) are preferred, because the resulting isoprene oligomerhas a large effect of improving low-heat-build-up properties, wet gripperformance, and abrasion resistance. Also, structures represented bythe formulas (g) to (q) are preferred, with structures represented bythe formulas (k), (l), and (q) being more preferred, because theresulting isoprene oligomer is excellent in antimicrobial properties.

(Process for Producing Isoprene Oligomer)

Examples of the process for producing the isoprene oligomer of thepresent invention include a process including performing biosynthesisfrom an allylic diphosphate and isopentenyl diphosphate, the allylicdiphosphate (hereinafter, also referred to as an allylic diphosphatederivative) being represented by the formula (3) shown below, wherein atleast one atom or group contained in isoprene units in the formula (3)is replaced by another atom or group. In the present specification, theallylic diphosphate derivative has three hydroxy groups bonded to aphosphorus atom, and some (or one) or all of these hydroxy groups aredissociated in an aqueous solution (for example, a group represented bythe formula (5) is then formed). In the present specification, theallylic diphosphate derivative also conceptually encompasses such groupsin which some (or one) or all of the hydroxy groups have beendissociated.

wherein p represents an integer of 1 to 10.

In the formula (3), p represents an integer of 1 to 10 (preferably 1 to4, more preferably 1 to 3).

In the present specification, examples of the atom or group (atom orgroup before replacement) contained in isoprene units in the formula (3)include the same as those exemplified as the atom or group (atom orgroup before replacement) contained in the trans structural moiety inthe formula (1).

In the present specification, examples of the another atom or group forreplacing the atom or group contained in isoprene units in the formula(3) include the same as those exemplified as the another atom or theanother group described for the formula (1).

For the replacement, it is preferable that the structure of moiety I inthe formula (I) should be maintained after the replacement, as describedabove. Specifically, it is preferable that at least one atom or groupcontained in moiety IV in the formula (3-1) shown below should bereplaced while no atom or group contained in moiety V in the formula(3-1) should be replaced. This allows favorable production of isopreneoligomers using the naturally occurring oligomer-producing enzymeprenyltransferase or any enzyme obtained by partial mutation thereof.

wherein p is as defined for p in the formula (3).

Specific examples of the allylic diphosphate derivative includecompounds represented by the following formulas (A) to (S).

In the present specification, OPP has three hydroxy groups bonded to aphosphorus atom, and some (or one) or all of these hydroxy groups aredissociated in an aqueous solution (for example, a group represented bythe formula (5) is then formed). In the present specification, OPP alsoconceptually encompasses such groups in which some (or one) or all ofthe hydroxy groups have been dissociated.

Allylic diphosphate derivatives represented by the formulas (A) to (S)and the like can be produced by those skilled in the art with referenceto the processes described in Examples, using, for example,dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate,geranylgeranyl diphosphate, geraniol, farnesol, or geranylgeraniol.

Examples of the process for biosynthesizing the isoprene oligomer of thepresent invention from the allylic diphosphate derivative andisopentenyl diphosphate include a process using an enzyme havingprenyltransferase activity. Specifically, the allylic diphosphatederivative and isopentenyl diphosphate may be allowed to react with eachother in the presence of an enzyme having prenyltransferase activity.

In the present specification, the enzyme having prenyltransferaseactivity means an enzyme having activity of catalyzing the condensationreaction between an allylic substrate (allylic diphosphate) andisopentenyl diphosphate to synthesize a new allylic diphosphate in whichone isoprene unit is added, and catalyzing a reaction through whichisopentenyl diphosphate is sequentially linked in a Z form (newly addedisoprene unit has a cis structure) starting from the allylic substrate(allylic diphosphate). Examples thereof include enzymes for catalyzingthe following reaction:

The presence of many such enzymes having prenyltransferase activity hasalready been confirmed (e.g., Z-nonaprenyl diphosphate synthase (Ishii,K. et al., (1986) Biochem, J., 233, 773) and undecaprenyl diphosphate(UPP) synthase (Takahashi, I. and Ogura, K. (1982) J. Biochem., 92,1527; and Keenman, M. V. and Allen, C. M. (1974) Arch. Biochem.Biophys., 161, 375)). Since the maximum number of isoprene units (m inthe formula (1)) each enzyme can produce is predetermined, the enzyme tobe used can be changed according to the number of isoprene units (m inthe formula (1)) of interest.

Examples of organisms containing the enzyme having prenyltransferaseactivity include Micrococcus luteus B-P 26, Escherichia coli,Saccharomyces cerevisiae, Arabidopsis thaliana, Hevea brasiliensis,Periploca sepium, Bacillus Stearothermophilus, and Sulfolobusacidocaldarius (ATCC49426).

The isoprene oligomer of the present invention can be obtained byreacting the allylic diphosphate derivative with isopentenyl diphosphatein the presence of the enzyme having prenyltransferase activity. Thephrase “in the presence of the enzyme having prenyltransferase activity”means the presence of a culture of the organism, biological cellsseparated from the culture, a treated product of the biological cells,the enzyme purified from the culture or the biological cells, a cultureof biological cells (transformant) transformed by a genetic engineeringapproach so as to express the enzyme having prenyltransferase activity(this enzyme also encompasses variant enzymes described later),biological cells separated from the culture, a treated product of thebiological cells, the enzyme purified from the culture or the biologicalcells, or the like.

In this context, the biological cells transformed so as to express theenzyme having prenyltransferase activity refer to a transformantprepared by a genetic engineering approach conventionally known in theart. The preparation method will be described later.

In order to obtain biological cells of the organism, the organism can becultured in an appropriate medium. The medium for this purpose is notparticularly limited as long as the organism can proliferate therein. Ausual medium containing usual carbon source, nitrogen source andinorganic ions, and optionally organic nutrient sources can be used.

For example, any carbon sources that can be utilized by the organism canbe used. Specific examples of usable carbon sources include: sugars suchas glucose, fructose, maltose, and amylose; alcohols such as sorbitol,ethanol, and glycerol; organic acids such as fumaric acid, citric acid,acetic acid, and propionic acid, and salts thereof; carbohydrates suchas paraffin; and mixtures thereof.

Examples of usable nitrogen sources include: inorganic ammonium saltssuch as ammonium sulfate and ammonium chloride; ammonium salts oforganic acids such as ammonium fumarate and ammonium citrate; nitratessuch as sodium nitrate and potassium nitrate; organic nitrogen compoundssuch as peptone, yeast extracts, meat extracts, and corn steep liquor;and mixtures thereof.

In addition, nutrient sources used in usual media, such as inorganicsalts, trace metal salts, vitamins, and hormones, can be mixedappropriately for use.

The culture conditions are not particularly limited and culture can beperformed at pH and temperature appropriately controlled in the rangesof pH 5 to 8 and temperature 20 to 60° C., for approximately 12 to 480hours under aerobic conditions.

Examples of the culture of the organism include a culture solutionobtained by culturing the organism under the culture conditionsdescribed above, and a culture filtrate (culture supernatant) obtainedby separating the organism (biological cells) from the culture solutionby filtration or the like. Also, examples of the biological cellsseparated from the culture include biological cells (organism) separatedfrom the culture solution by filtration, centrifugation, or the like.

Examples of the treated product of the biological cells includedisrupted biological cells obtained by homogenizing the biological cellsseparated from the culture, and disrupted biological cells obtained bysonicating the biological cells separated from the culture.

The enzyme purified from the culture or the biological cells may beobtained, for example, by a known purification operation, such assalting out, ion-exchange chromatography, affinity chromatography, orgel-filtration chromatography, for the enzyme present in the culture orthe biological cells. The purified enzyme is not particularly limited inits purity.

As described above, the isoprene oligomer of the present invention canbe obtained by reacting the allylic diphosphate derivative withisopentenyl diphosphate in the presence of the enzyme havingprenyltransferase activity. Specifically, for example, the culture ofthe biological cells, the purified enzyme or the like can be added intoa solution containing the allylic diphosphate derivative and isopentenyldiphosphate to perform the reaction. The reaction temperature can be setto, for example, 20 to 60° C.; the reaction time can be set to, forexample, 1 to 16 hours; and the pH can be set to, for example, 5 to 8.If necessary, magnesium chloride, a surfactant, 2-mercaptoethanol, andthe like may further be added thereto.

The isoprene oligomer of the present invention obtained by the reactionis usually represented by the formula (1) wherein Y is a grouprepresented by the formula (2) or a hydroxy group. This hydroxy group isformed through the hydrolysis of the group represented by the formula(2).

Further, the isoprene oligomer represented by the formula (1) wherein Yis a formyl group can be obtained, for example, by the oxidation of theisoprene oligomer of the formula (1) wherein Y is a group represented bythe formula (2).

Further, the isoprene oligomer represented by the formula (1) wherein Yis a carboxy group can be obtained, for example, by the oxidation of theisoprene oligomer of the formula (1) wherein Y is a group represented bythe formula (2).

Further, the isoprene oligomer represented by the formula (1) wherein Yis an ester group can be obtained, for example, by the oxidation andesterification of the isoprene oligomer of the formula (1) wherein Y isa group represented by the formula (2).

Further, the isoprene oligomer represented by the formula (1) wherein Yis a carbonyl group can be obtained, for example, by the oxidation andesterification of the isoprene oligomer of the formula (1) wherein Y isa group represented by the formula (2).

The isoprene oligomer of the present invention is obtained bybiosynthesis except for the organic synthesis of the allylic diphosphatederivative as an initiating substrate, and can thus take into accountthe exhaustion of petroleum resources and environmental issues.

(Enzyme Having Prenyltransferase Activity)

Next, the enzyme having prenyltransferase activity will be described.

As an example of the enzyme having prenyltransferase activity derivedfrom the organism, the base sequence and the amino acid sequence ofMicrococcus luteus B-P 26 (available from Dr. L. Jeffries, Walton OaksExperimental Station Vitamins, Ltd.)-derived undecaprenyl diphosphatesynthase are shown in SEQ ID NOs: 1 and 2, respectively, in the SequenceListing. The base sequence and the amino acid sequence of Micrococcusluteus B-P 26-derived undecaprenyl diphosphate synthase are known in theart and included in the database DDBJ (DNA Data Bank of Japan)(Accession No. AB004319 (base sequence) and Accession No. BAA31993.1(amino acid sequence)).

The original substrate (initiating substrate) for the enzyme (enzymehaving prenyltransferase activity) derived from this organism is anallylic diphosphate. Then, the allylic diphosphate derivative used as aninitiating substrate in the present invention originally functions as aninhibitor of the enzyme produced by the organism. Thus, the enzymeproduced by the organism often has low enzymatic activity on the allylicdiphosphate derivative (particularly, the compound represented by any ofthe formulas (G) to (Q)). Therefore, in the present invention, it ispreferable to use a variant enzyme having enhanced enzymatic activity onthe allylic diphosphate derivative.

In the case of using the variant enzyme, biological cells (transformant)transformed by a genetic engineering approach so as to express thevariant enzyme can be prepared.

The present inventors have prepared variant enzymes of the Micrococcusluteus B-P 26-derived undecaprenyl diphosphate synthase and successfullyenhanced the enzymatic activity on the allylic diphosphate derivative.

The three-dimensional structure of the Micrococcus luteus B-P 26-derivedundecaprenyl diphosphate synthase has already been known in the art(database PDB (RCSB Protein Data Bank), ID: 1f75). Thus, the presentinventors have conducted docking simulation based on thisthree-dimensional structure information and prepared variant enzymes onthe basis of the design philosophy that the enzymatic activity on theallylic diphosphate derivative can be enhanced by mutation of an aminoacid positioned in the vicinity of the hydrocarbon moiety around thediphosphate moiety of the allylic diphosphate so as to change the lengthof the side chain or the charge. Specifically, it is preferable toperform any of the following mutations (1) to (4):

(1) Substitution of asparagine at position 31 by alanine, glutamine,glycine, or aspartic acid;

(2) Substitution of leucine at position 91 by asparagine, aspartic acid,glycine, or lysine;

(3) Substitution of asparagine at position 77 by alanine, glutamine,glycine, or aspartic acid;

(4) Substitution of phenylalanine at position 95 by alanine, tryptophan,glycine, aspartic acid, or arginine.

Specifically, the following variant enzymes were prepared:

variant enzyme N31A: asparagine at position 31 was substituted byalanine (its base sequence and amino acid sequence are shown in SEQ IDNOs: 3 and 4, respectively, in the Sequence Listing);

variant enzyme N77A: asparagine at position 77 was substituted byalanine (its base sequence and amino acid sequence are shown in SEQ IDNOs: 5 and 6, respectively, in the Sequence Listing);

variant enzyme L91N: leucine at position 91 was substituted byasparagine (its base sequence and amino acid sequence are shown in SEQID NOs: 7 and 8, respectively, in the Sequence Listing);

variant enzyme L91D: leucine at position 91 was substituted by asparticacid (its base sequence and amino acid sequence are shown in SEQ ID NOs:9 and 10, respectively, in the Sequence Listing);

variant enzyme N31Q: asparagine at position 31 was substituted byglutamine (its base sequence and amino acid sequence are shown in SEQ IDNOs: 11 and 12, respectively, in the Sequence Listing);

variant enzyme N77Q: asparagine at position 77 was substituted byglutamine (its base sequence and amino acid sequence are shown in SEQ IDNOs: 13 and 14, respectively, in the Sequence Listing);

variant enzyme L91G: leucine at position 91 was substituted by glycine(its base sequence and amino acid sequence are shown in SEQ ID NOs: 15and 16, respectively, in the Sequence Listing);

variant enzyme L91K: leucine at position 91 was substituted by lysine(its base sequence and amino acid sequence are shown in SEQ ID NOs: 17and 18, respectively, in the Sequence Listing);

variant enzyme F95A: phenylalanine at position 95 was substituted byalanine (its base sequence and amino acid sequence are shown in SEQ IDNOs: 19 and 20, respectively, in the Sequence Listing);

variant enzyme F95W: phenylalanine at position 95 was substituted bytryptophan (its base sequence and amino acid sequence are shown in SEQID NOs: 21 and 22, respectively, in the Sequence Listing).

Among them, variant enzyme N77A, variant enzyme L91D, and variant enzymeL91K are preferred.

Although the use of the Micrococcus luteus B-P 26-derived undecaprenyldiphosphate synthase as a wild-type enzyme is described herein, variantenzymes can be prepared by similar approaches using other enzymes havingprenyltransferase activity as wild-type enzymes.

Specific examples of the enzyme having prenyltransferase activityinclude the following protein [1]:

[1] a protein having an amino acid sequence represented by any of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22.

It is also known that enzymes having an amino acid sequence derived fromthe original amino acid sequence by substitution, deletion, insertion,or addition of one or more amino acids may have enzymatic activity.Thus, specific examples of the enzyme having prenyltransferase activityalso include the following protein [2]:

[2] a protein having an amino acid sequence derived from the amino acidsequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, and 22 by substitution, deletion, insertion, or addition of oneor more amino acids, and having activity of catalyzing a reactionbetween an allylic diphosphate (allylic diphosphate derivative) andisopentenyl diphosphate, the allylic diphosphate being represented bythe following formula (3), wherein at least one atom or group containedin isoprene units in the formula (3) is replaced by another atom orgroup:

wherein p represents an integer of 1 to 10.

For maintaining the activity of catalyzing the reaction between theallylic diphosphate derivative and isopentenyl diphosphate, it ispreferable that the amino acid sequence should contain substitution,deletion, insertion, or addition of preferably one or more amino acids,more preferably 1 to 100 amino acids, even more preferably 1 to 75 aminoacids, particularly preferably 1 to 50 amino acids, further preferably 1to 25 amino acids, further preferably 1 to 12 amino acids, furtherpreferably 1 to 5 amino acids, and most preferably 1 to 3 amino acids.

It is also known that proteins having an amino acid sequence having highsequence identity to the amino acid sequence of the enzyme havingprenyltransferase activity may have similar activity. Thus, specificexamples of the enzyme having prenyltransferase activity also includethe following protein [3]:

[3] a protein having an amino acid sequence having 45% or highersequence identity to the amino acid sequence represented by any of SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, and having activityof catalyzing a reaction between the allylic diphosphate derivative andisopentenyl diphosphate.

For maintaining the activity of catalyzing the reaction between theallylic diphosphate derivative and isopentenyl diphosphate, the sequenceidentity to the amino acid sequence represented by any of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 is preferably 45% or higher,more preferably 60% or higher, even more preferably 70% or higher,particularly preferably 80% or higher, further preferably 90% or higher,further preferably 95% or higher, further preferably 98% or higher, andmost preferably 99% or higher.

The amino acid sequence or base sequence identity can be determinedusing the algorithm BLAST by Karlin and Altschul [Pro. Natl. Acad. Sci.USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)].

Examples of the method for confirming that a protein has activity ofcatalyzing the reaction between the allylic diphosphate derivative andisopentenyl diphosphate include a method involving preparing atransformant expressing the protein by a method conventionally known inthe art, producing the protein using the transformant, and thenquantitatively or qualitatively analyzing the substrate or product byHPLC (high-performance liquid chromatography), TLC (thin-layerchromatography), or the like to confirm whether or not the protein cancatalyze the reaction between the allylic diphosphate derivative andisopentenyl diphosphate.

(DNA Encoding Enzyme Having Prenyltransferase Activity)

Examples of DNA encoding the enzyme having prenyltransferase activityinclude the following DNAs [1] to [3]:

[1] DNA encoding any of the proteins [1] to [3];

[2] DNA having a base sequence represented by any of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, and 21; and

[3] DNA hybridizing under stringent conditions to DNA having a basesequence complementary to the base sequence represented by any of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21; and encoding a proteinhaving activity of catalyzing a reaction between the allylic diphosphatederivative and isopentenyl diphosphate.

In this context, the term “hybridizing” means a step in which the DNAhybridizes to DNA having a specific base sequence or a portion of theDNA. Thus, the base sequence of the DNA having a specific base sequenceor the portion of the DNA may be DNA of length that is useful as a probein Northern or Southern blot analysis or can be used as anoligonucleotide primer in PCR (polymerase chain reaction) analysis.Examples of the DNA used as a probe include DNA of at least 100 bases orlonger, preferably of 200 bases or longer, and more preferably of 500bases or longer. DNA of at least 10 bases or longer, preferably of 15bases or longer, may be used.

Methods for DNA hybridization experiment are well known. Hybridizationconditions can be determined according to the description of, forexample, Molecular Cloning, 2nd ed. and 3rd ed. (2001), Methods forGeneral and Molecular Bacteriology, ASM Press (1994), or Immunologymethods manual, Academic press (Molecular) as well as many otherstandard textbooks to conduct the experiment.

Examples of the stringent conditions include conditions involving, forexample, incubating a DNA-immobilized filter and probe DNA overnight at42° C. in a solution containing 50% formamide, 5×SSC (750 mM sodiumchloride and 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/l denatured salmonsperm DNA, and then washing the filter in, for example, a 0.2×SSCsolution at approximately 65° C. Lower stringent conditions may be used.The stringent conditions can be changed by adjustment of theconcentration of formamide (lower concentration of formamide gives lowerstringent conditions) and change in salt concentration and temperatureconditions. Examples of low stringent conditions include conditionsinvolving, for example, overnight incubation at 37° C. in a solutioncontaining 6×SSCE (20×SSCE corresponds to 3 mol/l sodium chloride, 0.2mol/l sodium dihydrogen phosphate, and 0.02 mol/l EDTA, pH 7.4), 0.5%SDS, 30% formamide, and 100 μg/l denatured salmon sperm DNA, followed bywashing using a solution of 1×SSC and 0.1% SDS at 50° C. Also, examplesof lower stringent conditions include conditions involving hybridizationusing a solution with a higher salt concentration (e.g., 5×SSC) underthe low stringent conditions, followed by washing.

These various conditions may be set by the addition or change of ablocking reagent used for suppressing background in the hybridizationexperiment. The addition of the blocking reagent may be accompanied bychange of the hybridization conditions in order to adapt the conditionsto each experiment.

Examples of DNA capable of hybridization under the stringent conditionsdescribed above include DNA having a base sequence having at least 80%or higher, preferably 90% or higher, more preferably 95% or higher, evenmore preferably 98% or higher, particularly preferably 99% or highersequence identity to the base sequence represented by any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 when calculated on the basisof the parameters using a program such as BLAST and FASTA.

Examples of the method for confirming that the DNA hybridizing understringent conditions to the DNA encodes a protein having activity ofcatalyzing the reaction between the allylic diphosphate derivative andisopentenyl diphosphate include a method involving preparing recombinantDNA containing the DNA by a method conventionally known in the art,introducing the recombinant DNA into host cells, culturing the obtainedbiological cells, purifying the protein from the obtained culture, andthen quantitatively or qualitatively analyzing the substrate or productby HPLC, TLC, or the like to confirm whether or not the protein cancatalyze the reaction between the allylic diphosphate derivative andisopentenyl diphosphate.

Each variant enzyme and DNA encoding the variant enzyme can be obtainedby the site-directed mutagenesis of, for example, the base sequencerepresented by SEQ ID NO: 1 (base sequence of Micrococcus luteus B-P26-derived undecaprenyl diphosphate synthase) using a site-directedmutagenesis method described in Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989), CurrentProtocols in Molecular Biology, John Wiley & Sons (1987-1997), NucleicAcids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409(1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985),Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc.

(Transformant)

Next, the method for preparing biological cells (transformant)transformed so as to express the enzyme having prenyltransferaseactivity will be described briefly. Here, mainly, a method for preparinga transformant that has been transformed so as to express the variantenzyme will be described briefly. Such a transformant can be prepared bya method conventionally known in the art as long as the designphilosophy is determined.

For the mutagenesis, first, primers are designed so that they allowmutagenesis at a site of interest. Examples of the base sequences of theprimers include base sequences described in Examples (see SEQ ID NOs: 23to 42 in the Sequence Listing). Next, for example, mutagenized linearDNA is amplified by the PCR method or the like using DNA containing thebase sequence represented by SEQ ID NO: 1 (base sequence of Micrococcusluteus B-P 26-derived undecaprenyl diphosphate synthase) as template DNAwith the primers described above. Then, the obtained linear DNA isinserted downstream of a promoter in an appropriate expression vectorusing appropriate restriction enzymes or the like to prepare recombinantDNA. Then, the recombinant DNA is introduced into host cells compatiblewith the expression vector, whereby a transformant can be obtained.

Alternatively, for example, DNA containing the base sequence representedby SEQ ID NO: 1 (base sequence of Micrococcus luteus B-P 26-derivedundecaprenyl diphosphate synthase) is inserted downstream of a promoterin an appropriate expression vector using appropriate restrictionenzymes or the like. Mutagenized DNA, which is obtained by the PCRmethod or the like using the expression vector as template DNA with theprimers described above, is rendered cyclic by polymerase to preparerecombinant DNA. Then, the recombinant DNA is introduced into host cellscompatible with the expression vector, whereby a transformant can beobtained.

When such mutagenesis is unnecessary, for example, DNA containing thebase sequence represented by SEQ ID NO: 1 (base sequence of Micrococcusluteus B-P 26-derived undecaprenyl diphosphate synthase) is inserteddownstream of a promoter in an appropriate expression vector usingappropriate restriction enzymes or the like to prepare recombinant DNA.Then, the recombinant DNA is introduced into host cells compatible withthe expression vector, whereby a transformant can be obtained.

Although the description above is about the case using the DNAcontaining the known base sequence represented by SEQ ID NO: 1 (basesequence of Micrococcus luteus B-P 26-derived undecaprenyl diphosphatesynthase), DNA encoding any of other prenyltransferase activity-havingenzymes derived from the organism or prenyltransferase activity-havingenzymes derived from organisms other than the organism may be used. Inthis case, screening can be performed by a known approach using, forexample, a portion of the base sequence represented by SEQ ID NO: 1 as aprobe to identify DNA encoding the enzyme having prenyltransferaseactivity, followed by isolation. The method for isolating the DNAmolecule of interest using the DNA molecule as a probe is described in,for example, Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

Alternatively, the following method may be used: the organism-derivedenzyme having prenyltransferase activity is purified by the purificationoperation as described above, and the amino acid sequence of thepurified enzyme is determined to identify DNA encoding the enzyme,followed by isolation.

Any of microorganisms, yeasts, animal cells, insect cells, plant cells,and the like can be used as host cells as long as they are capable ofexpressing the gene of interest.

Examples of usable expression vectors include those which are capable ofautonomously replicating in the host cells or being incorporated intothe chromosome and contain a promoter at a position that allowstranscription of the recombinant DNA.

When a prokaryotic organism such as bacteria is used as host cells, itis preferable that the recombinant DNA should be recombinant DNA that iscapable of autonomously replicating in the prokaryotic organism and isalso composed of a promoter, a ribosomal binding sequence, DNA encodingthe enzyme having prenyltransferase activity, and a transcriptiontermination sequence. The recombinant DNA may further contain a genecontrolling the promoter.

Examples of the expression vector include pCold I (manufactured byTakara Bio Inc.), pCDF-1b and pRSF-1b (all manufactured by Novagen),pMAL-c2x (manufactured by New England Biolabs Inc.), pGEX-4T-1(manufactured by GE Healthcare Biosciences Inc.), pTrcHis (manufacturedby Invitrogen Corp.), pSE280 (manufactured by Invitrogen Corp.),pGEMEX-1 (manufactured by Promega Corp.), pQE-30 (manufactured byQiagen), pET-3 to pET-52 (manufactured by Novagen), pKYP10 (JP 58-110600A), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol.Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306(1985)], pBluescript II SK(+) and pBluescript II KS(−) (manufactured byStratagene Corp.), pTrS30 [prepared from Escherichia coli JM109/pTrS30(FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109/pTrS32(FERM BP-5408)], pPAC31 (WO98/12343), pUC19 [Gene, 33, 103 (1985)],pSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by TakaraBio Inc.), and pPA1 (JP 63-233798 A).

Any promoter that functions in host cells such as Escherichia coli canbe used. Examples thereof include: promoters derived from E. coli,phages, etc., such as trp promoter (P_(trp)), T7 promoter, lac promoter(P_(lac)), P_(L) promoter, P_(R) promoter, and P_(SE) promoter; and SPO1promoter, SPO2 promoter, and penP promoter. Also, for example,artificially designed or modified promoters such as a promoter havingtwo P_(trp) sequences arranged in series, tac promoter, lacT7 promoter,and let I promoter, may be used.

Furthermore, xylA promoter for expression in microorganisms belonging tothe genus Bacillus [Appl. Microbiol. Biotechnol., 35, 594-599 (1991)],P54-6 promoter for expression in microorganisms belonging to the genusCorynebacterium [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)], andthe like may be used.

It is preferable to use a plasmid having an appropriately adjusteddistance (e.g., 6 to 18 bases) between a Shine-Dalgarno sequence(ribosomal binding sequence) and an initiation codon.

Examples of the prokaryotic organism include microorganisms belonging tothe genera Escherichia, Serratia, Bacillus, Brevibacterium,Corynebacterium, Microbacterium, Pseudomonas, Agrobacterium,Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azotobacter,Chromatium, Erwinia, Methylobacterium, Phormidium, Rhodobacter,Rhodopseudomonas, Rhodospirillum, Scenedesmus, Streptomyces,Synechoccus, and Zymomonas, for example, Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α,Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coliW1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coliNo. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coliMP347, Escherichia coli NM522, Escherichia coli BL21, Bacillus subtilisATCC33712, Bacillus megaterium, Brevibacterium ammoniagenes,Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticumATCC14066, Brevibacterium flavum ATCC14067, Brevibacteriumlactofermentum ATCC13869, Corynebacterium glutamicum ATCC13032,Corynebacterium glutamicum ATCC14297, Corynebacterium acetoacidophilumATCC13870, Microbacterium ammoniaphilum ATCC15354, Serratia ficaria,Serratia fonticola, Serratia liquefaciens, Serratia marcescens,Pseudomonas sp. D-0110, Agrobacterium radiobacter, Agrobacteriumrhizogenes, Agrobacterium rubi, Anabaena cylindrica, Anabaena doliolum,Anabaena flos-aquae, Arthrobacter aurescens, Arthrobacter citreus,Arthrobacter globiformis, Arthrobacter hydrocarboglutamicus,Arthrobacter mysorens, Arthrobacter nicotianae, Arthrobacterparaffineus, Arthrobacter protophormiae, Arthrobacter roseoparaffinus,Arthrobacter sulfureus, Arthrobacter ureafaciens, Chromatium buderi,Chromatium tepidum, Chromatium vinosum, Chromatium warmingii, Chromatiumfluviatile, Erwinia uredovora, Erwinia carotovora, Erwinia ananas,Erwinia herbicola, Erwinia punctata, Erwinia terreus, Methylobacteriumrhodesianum, Methylobacterium extorquens, Phormidium sp. ATCC29409,Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodopseudomonasblastica, Rhodopseudomonas marina, Rhodopseudomonas palustris,Rhodospirillum rubrum, Rhodospirillum salexigens, Rhodospirillumsalinarum, Streptomyces ambofaciens, Streptomyces aureofaciens,Streptomyces aureus, Streptomyces fungicidicus, Streptomycesgriseochromogenes, Streptomyces griseus, Streptomyces lividans,Streptomyces olivogriseus, Streptomyces rameus, Streptomycestanashiensis, Streptomyces vinaceus, and Zymomonas mobilis.

The recombinant DNA can be introduced by any method for introducing DNAinto such host cells. Examples thereof include a method using calciumions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method(JP 63-248394 A), an electroporation method [Nucleic Acids Res., 16,6127 (1988)], and a heat shock method.

When a yeast strain is used as host cells, YEp13 (ATCC37115), YEp24(ATCC37051), YCp50 (ATCC37419), pHS19, or pHS15, for example, can beused as an expression vector.

In this case, any promoter that functions in the yeast strain can beused. Examples thereof include promoters such as PHO5 promoter, PGKpromoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter,heat-shock polypeptide promoter, MFα1 promoter, and CUP 1 promoter.

Examples of the host cells include yeast strains belonging to the generaSaccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon,Schwanniomyces, Pichia, Candida and the like and specifically includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichiapastoris, and Candida utilis.

The recombinant DNA can be introduced by any method for introducing DNAinto yeast. Examples thereof include an electroporation method [MethodsEnzymol., 194, 182 (1990)], a spheroplast method [Proc. Natl. Acad.Sci., USA, 81, 4889 (1984)], and a lithium acetate method [J.Bacteriol., 153, 163 (1983)].

When animal cells are used as a host, pcDNAI, pcDM8 (commerciallyavailable from Funakoshi Corp.), pAGE107 (JP 3-22979 A), pAS3-3 (JP2-227075 A), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufacturedby Invitrogen Corp.), pREP4 (manufactured by Invitrogen Corp.), pAGE103[J. Biochem, 101, 1307 (1987)], pAGE210, pAMo, or pAMoA, for example,can be used as an expression vector.

In this case, any promoter that functions in the animal cells can beused. Examples thereof include cytomegalovirus (CMV) IE (immediateearly) gene promoter, SV40 early promoter, metallothionein promoter,retrovirus promoter, heat-shock promoter, and SRα promoter. Thesepromoters may be used in combination with human CMV IE gene enhancer.

Examples of the host cells include mouse myeloma cells, rat myelomacells, mouse hybridoma cells, human Namalwa cells or Namalwa KJM-1cells, human embryonic kidney cells, human leukemia cells, African greenmonkey kidney cells, Chinese hamster ovary (CHO) cells, and HBT5637 (JP63-299 A).

Specific examples thereof include: mouse myeloma cells such as SP2/0 andNSO; rat myeloma cells such as YB2/0; human embryonic kidney cells suchas HEK293 (ATCC CRL-1573); human leukemia cells such as BALL-1; andAfrican green monkey kidney cells such as COS-1 and COS-7.

The recombinant DNA can be introduced by any method for introducing DNAinto animal cells. Examples thereof include an electroporation method[Cytotechnology, 3, 133 (1990)], a calcium phosphate method (JP 2-227075A), a lipofection method [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)],and a method described in Virology, 52, 456 (1973).

When insect cells are used as a host, the protein can be produced by amethod described in, for example, Baculovirus Expression Vectors, ALaboratory Manual, W. H. Freeman and Company, New York (1992), CurrentProtocols in Molecular Biology, or Molecular Biology, A LaboratoryManual, Bio/Technology, 6, 47 (1988).

Specifically, insect cells are cotransfected with a vector fortransferring the recombinant gene and baculovirus, so that recombinantvirus is obtained in the culture supernatant of the insect cells. Then,insect cells are further infected with the recombinant virus, wherebythe protein can be produced.

Examples of the gene transfer vector used in this method includepVL1392, pVL1393, and pBlueBac III (all manufactured by InvitrogenCorp.).

Examples of the baculovirus include Autographa californica nuclearpolyhedrosis virus, which infects insects of the family Noctuidae.

Examples of the insect cells include Spodoptera frugiperda ovary cells,Trichoplusia ni ovary cells, and silkworm ovary-derived cultured cells.

Specific examples thereof include: Spodoptera frugiperda ovary cellssuch as Sf9 and Sf21 (Baculovirus Expression Vectors: A LaboratoryManual); Trichoplusia ni ovary cells such as High 5 and BTI-TN-5B1-4(manufactured by Invitrogen Corp.); and silkworm ovary-derived culturedcells such as Bombyx mori N4.

Examples of the method for cotransfecting insect cells with the vectorfor transferring the recombinant gene and the baculovirus to prepare therecombinant virus include a calcium phosphate method (JP 2-227075 A) anda lipofection method [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)].

When plant cells are used as host cells, a Ti plasmid or tobacco mosaicvirus vector, for example, can be used as an expression vector.

In this case, any promoter that functions in the plant cells can beused. Examples thereof include cauliflower mosaic virus (CaMV) 35Spromoter and rice actin-1 promoter.

Examples of the host cells include cells of plants such as tobacco,potato, tomato, carrot, soybean, oilseed rape, alfalfa, rice, wheat, andbarley.

The recombinant vector can be introduced by any method for introducingDNA into plant cells. Examples thereof include a method usingAgrobacterium (JP 59-140885 A, JP 60-70080 A, and WO94/00977), anelectroporation method (JP 60-251887 A), and a method using a particlegun (gene gun) (Japanese Patent Nos. 2606856 and 2517813).

The host may be any of microorganisms, yeasts, animal cells, insectcells, plant cells, and the like and may be preferably any ofmicroorganisms, more preferably microorganisms belonging to the genusEscherichia, and even more preferably microorganisms belonging toEscherichia coli.

When expressed by yeasts, animal cells, insect cells, and plant cells,sugar- or sugar chain-added proteins can be obtained.

The obtained transformant is cultured in a medium and the enzyme havingprenyltransferase activity is then generated and accumulated in theculture, whereby the enzyme having prenyltransferase activity can beproduced. If necessary, the enzyme may be purified by the purificationoperation described above.

The transformant can be cultured in a medium according to a usual methodused for culturing host cells. For example, the culture can be performedin the composition of the medium and the culture conditions describedabove.

(Polyisoprene)

Next, the polyisoprene of the present invention will be described. Thepolyisoprene of the present invention contains a trans structural moietyand a cis structural moiety as represented by the formula (4) shownbelow, wherein at least one atom or group contained in the transstructural moiety is replaced by another atom or group. In this context,the trans structural moiety means a moiety of repeated isoprene unitswith a trans structure (moiety C in the formula (4) shown below). Also,the cis structural moiety means a moiety of repeated isoprene units witha cis structure (moiety ( )_(q) (moiety D) in the formula (4) shownbelow).

wherein n represents an integer of 1 to 10; q represents an integer of30 to 40000; and Y represents a hydroxy group, a formyl group, a carboxygroup, an ester group, a carbonyl group, or a group represented by thefollowing formula (2).

The polyisoprene of the present invention is structurally similar tonatural rubber and is highly compatible with rubber molecules. Moreover,the polyisoprene of the present invention has been modifiedsubstantially only at a terminal site of the molecule. Specifically, thepolyisoprene of the present invention contains a hydroxy group, a formylgroup, a carboxy group, an ester group, a carbonyl group, or a grouprepresented by the formula (2), positioned at the end of the cisstructural moiety, and at least one atom or group contained in the transstructural moiety is replaced by another atom or group. Thus, thepolyisoprene of the present invention strongly interacts with fillersuch as silica while the original properties of polyisoprene are notimpaired. Since the polyisoprene of the present invention is highlycompatible with rubber and strongly interacts with filler such as silicaas described above, a rubber composition containing this polyisoprene isobtained as a rubber composition in which rubber molecules are combinedwith filler in a level higher than ever. The resulting rubbercomposition can have, for example, improved low-heat-build-upproperties, wet grip performance, and abrasion resistance.

The polyisoprene of the present invention contains a polar group or thelike only at the terminal site of the cis structural moiety and a siteclose to the end of the trans structural moiety. The polyisoprenetherefore offers high dispersibility of filler such as silica and has alarge effect of improving, for example, low-heat-build-up properties,wet grip performance, and abrasion resistance while the originalproperties of polyisoprene are not impaired, compared with polyisoprenescontaining a polar group or the like in the main chain moiety or only atthe terminal site of the cis structural moiety.

In the formula (4), n is as defined for n in the formula (1).

In the formula (4), q represents an integer of 30 to 40000 (preferably15000 to 30000, more preferably 15000 to 20000).

In the formula (4), Y is as defined for Y in the formula (1). Here, Y ispreferably a hydroxy group or a carboxy group, because the resultingpolyisoprene strongly interacts with filler such as silica.

Examples of the atom or group (atom or group before replacement)contained in the trans structural moiety include the same as thoseexemplified as the atom or group (atom or group before replacement)contained in the trans structural moiety in the formula (1).

Examples of the another atom or group include the same as thoseexemplified as the another atom or the another group described about theformula (1).

As described above, at least one atom or group contained in the transstructural moiety is replaced by another atom or group. For thereplacement, it is preferable, as described for the isoprene oligomer,that at least one atom or group contained in moiety VI in the followingformula (4-1) should be replaced while no atom or group contained inmoiety VII in the formula (4-1) should be replaced:

wherein n, q, and Y are as defined for n, q, and Y in the formula (4).

Specific examples of the trans structural moiety in the formula (4)include structures represented by the formulas (a) to (s) mentionedabove. Among them, structures represented by the formulas (c), (d), (e),(f), (k), (l), and (r) are preferred because the resulting polyisoprenemore strongly interacts with filler such as silica and has a largeeffect of improving low-heat-build-up properties, wet grip performance,and abrasion resistance.

(Process for Producing Polyisoprene)

Examples of the process for producing the polyisoprene of the presentinvention include a process including performing biosynthesis from theisoprene oligomer of the present invention and isopentenyl diphosphate.

The polyisoprene of the present invention is obtained by biosynthesisexcept for the organic synthesis of the allylic diphosphate derivativeas an initiating substrate, and can thus take into account theexhaustion of petroleum resources or environmental issues.

It has heretofore been known that natural rubber latex contains anenzyme, a rubber elongation factor, and the like having activity ofcatalyzing the condensation reaction between an isoprene oligomer andisopentenyl diphosphate and catalyzing a reaction as shown below throughwhich isopentenyl diphosphate is sequentially linked in a Z form (newlyadded isoprene unit has a cis structure) starting from the isopreneoligomer to produce a polyisoprene.

In the present invention, the polyisoprene can be produced using thisenzyme, rubber elongation factor, or the like.

Specifically, examples of the method for biosynthesizing thepolyisoprene of the present invention from the isoprene oligomer of thepresent invention and isopentenyl diphosphate include a method using theenzyme, the rubber elongation factor, or the like contained in naturalrubber latex. Alternatively, the enzyme, the rubber elongation factor,or the like cloned from natural rubber latex may be used.

Specifically, the isoprene oligomer of the present invention andisopentenyl diphosphate can be allowed to react with each other in thepresence of the enzyme and/or the rubber elongation factor. Morespecifically, for example, natural rubber latex or the enzyme, therubber elongation factor, or the like separated from the natural rubberlatex can be added into a solution containing the isoprene oligomer ofthe present invention and isopentenyl diphosphate to perform thereaction. The reaction temperature can be set to, for example, 20 to 40°C.; the reaction time can be set to, for example, 1 to 72 hours; and thepH can be set to, for example, 6 to 8. If necessary, magnesium chloride,a surfactant, 2-mercaptoethanol, and the like may further be addedthereto.

The polyisoprene of the present invention obtained by the reaction isusually represented by the formula (4) wherein Y is a group representedby the formula (2) or a hydroxy group. This hydroxy group is formedthrough the hydrolysis of the group represented by the formula (2).

Further, the polyisoprene represented by the formula (4) wherein Y is aformyl group can be obtained, for example, by the oxidation of thepolyisoprene of the formula (4) wherein Y is a group represented by theformula (2).

Further, the polyisoprene represented by the formula (4) wherein Y is acarboxy group can be obtained, for example, by the oxidation of thepolyisoprene of the formula (4) wherein Y is a group represented by theformula (2).

Further, the polyisoprene represented by the formula (4) wherein Y is anester group can be obtained, for example, by the oxidation andesterification of the polyisoprene of the formula (4) wherein Y is agroup represented by the formula (2).

Further, the polyisoprene represented by the formula (4) wherein Y is acarbonyl group can be obtained, for example, by the oxidation andesterification of the polyisoprene of the formula (4) wherein Y is agroup represented by the formula (2).

The natural rubber latex is not particularly limited in its origin.Examples thereof include Hevea brasiliensis, Ficus elastica, Ficuslyrata, Ficus benjamina, Ficus religiosa, Ficus benghalensis, andLactarius chrysorrheus. Among them, Hevea brasiliensis is preferredbecause it produces rubber having a large molecular weight and its latexcontains a large quantity of rubber.

The trunk of, for example, Hevea brasiliensis is cut in grooves (tapped)using a knife or the like, and natural rubber latex flowing out from thecleaved latex vessels is collected, whereby natural rubber latex can beobtained.

Examples of the enzyme and the rubber elongation factor separated fromthe natural rubber latex include a serum, a bottom fraction, and arubber fraction separated by centrifugation of natural rubber latex. Theserum, the bottom fraction, and the rubber fraction contain the enzyme,the rubber elongation factor, or the like.

(Rubber Composition)

The rubber composition of the present invention includes the isopreneoligomer of the present invention and/or the polyisoprene of the presentinvention. Accordingly, the rubber composition of the present inventionis excellent in low-heat-build-up properties, wet grip performance, andabrasion resistance. In this context, the polyisoprene of the presentinvention can be used as a rubber component.

The content of the polyisoprene of the present invention is preferably20% by mass or higher, more preferably 40% by mass or higher, and evenmore preferably 60% by mass or higher, with respect to 100% by mass ofrubber components, and may be 100% by mass.

Examples of rubber components that can be used in addition to thepolyisoprene of the present invention include diene rubbers such asisoprene rubber (IR), natural rubber (NR), butadiene rubber (BR),styrene-butadiene rubber (SBR), styrene-isoprene-butadiene rubber(SIBR), chloroprene rubber (CR), and acrylonitrile-butadiene rubber(NBR). These rubber components may be used alone or in combination oftwo or more thereof. Among them, NR and BR are preferred.

For allowing a rubber composition to contain the isoprene oligomer ofthe present invention, it is preferable to use NR as a rubber componentbecause it is highly compatible with isoprene oligomers. The combineduse of the isoprene oligomer of the present invention and NR is morefavorable for the effects of the addition of the isoprene oligomer ofthe present invention.

When the rubber composition contains the isoprene oligomer of thepresent invention, the content of NR is preferably 20% by mass orhigher, more preferably 40% by mass or higher, and even more preferably60% by mass or higher, with respect to 100% by mass of rubbercomponents, and may be 100% by mass.

The content of the isoprene oligomer of the present invention ispreferably 1 part by mass or higher, and more preferably 2 parts by massor higher, with respect to 100 parts by mass of rubber components. Ifthe content is less than 1 part by mass, the effects of the addition ofthe isoprene oligomer of the present invention might be achievedinsufficiently. Also, the content of the isoprene oligomer is preferably20 parts by mass or lower, and more preferably 15 parts by mass orlower. If the content exceeds 20 parts by mass, the strength and, byextension, abrasion resistance of the resulting rubber composition maybe reduced.

Examples of filler that can be used in the present invention includesilica, carbon black, clay, and calcium carbonate.

In the present invention, it is preferable to use silica as filler. Therubber composition further containing silica ensures sufficientachievement of the effects of the addition of the isoprene oligomer ofthe present invention and/or the polyisoprene of the present invention.Examples of the silica include, but not particularly limited to, drysilica (silicic anhydride) and wet silica (hydrous silicic acid). Wetsilica is preferred because of being rich in silanol groups.

For the present invention, it is also preferable to use carbon black asfiller. In this case as well, the effects of the addition of theisoprene oligomer of the present invention and/or the polyisoprene ofthe present invention can be achieved sufficiently.

The rubber composition of the present invention may appropriatelycontain, in addition to the components described above, compoundingingredients generally used in the production of rubber compositions, forexample, a silane coupling agent, zinc oxide, stearic acid, variousantioxidants, a softener (e.g., oil), wax, a vulcanizing agent (e.g.,sulfur), and a vulcanization accelerator.

The rubber composition of the present invention can be produced using aprocess known in the art and can be produced, for example, by a processinvolving kneading the components using a rubber kneading apparatus suchas an open roll mill or a Banbury mixer, followed by vulcanization.

The rubber composition of the present invention can be used suitably invarious tire components (e.g., treads, sidewalls, undertread, plies,breakers, and carcass) and the like.

(Pneumatic Tire)

The pneumatic tire of the present invention can be produced by a usualprocess using the rubber composition. Specifically, the unvulcanizedrubber composition is extruded and processed into a shape appropriatefor a tire component (e.g., treads and sidewalls), arranged by a usualmethod in a tire building machine, and assembled with other tirecomponents to form an unvulcanized tire. This unvulcanized tire isheated and pressurized in a vulcanizer, whereby a tire can be produced.

EXAMPLES

The present invention will be described more specifically with referenceto Examples. However, the present invention is not intended to belimited to these Examples.

Preparation of Initiating Substrate Production Example 1 Synthesis of10-acetyl-3,7-dimethyl-dodeca-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (S))

Synthesis was carried out with farnesol as a starting material. Thehydroxy group of farnesol was protected using imidazole andtert-butylphenylchlorosilane (TBDPS) in anhydrous dichloromethane toobtain a TBDPS-protected form (compound represented by (ai) below)(yield: 92%). The olefin at position 10 was oxidized usingm-chloroperbenzoic acid in anhydrous dichloromethane to obtain an epoxyform (compound represented by (aii) below) (yield: 9%). Next, the epoxywas oxidized with orthoperiodic acid in anhydrous tetrahydrofuran toobtain an aldehyde form (compound represented by (aiii) below) (yield:28%). Next, a Grignard reagent was prepared from magnesium and butanebromide in anhydrous methanol, and the aldehyde form was added to theGrignard reagent to obtain a secondary alcohol form (compoundrepresented by (aiv) below) (yield: 68%). The secondary hydroxy groupwas protected by acetylation with acetic anhydride in the presence ofdimethylaminopyridine in an anhydrous dichloromethane solvent to obtainan ester form (compound represented by (av) below) (yield: 81%). TheTBDPS protective group was deprotected using tetra-n-butylammoniumhydrate in anhydrous tetrahydrofuran to obtain a primary alcohol form(compound represented by (avi) below) (yield: 82%). Next, the primaryhydroxy group was replaced by chlorine using N-chlorosuccinimide anddimethyl sulfide in an anhydrous dichloromethane solvent at −40° C. orlower to obtain a chloride (compound represented by (avii) below)(yield: 82%). Next, the chloride was diphosphorylated usingtris-tetra-n-butylammonium hydrogen pyrophosphate in anhydrousacetonitrile to obtain a compound represented by (aviii) below (compoundrepresented by the formula (S)) as the substance of interest (yield:42%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 2 Synthesis of8-methoxy-3,7-dimethyl-dodeca-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (B))

Synthesis was carried out with geraniol as a starting material. Geraniolwas acetylated using pyridine and acetic anhydride in anhydrousdichloromethane to obtain an acetate (compound represented by (bi)below) (yield: 95%). Next, the carbon atom at position 8 was subjectedto selenium oxidation in ethanol to obtain an aldehyde form (compoundrepresented by (bii) below) (yield: 24%). Next, the aldehyde wasalkaline-hydrolyzed using potassium hydroxide to obtain an alcohol form(compound represented by (biii) below) (yield: 38%). Next, the alcoholwas treated with imidazole and tert-butyldiphenylsilyl chloride (TBDPS)in anhydrous dichloromethane to obtain a compound represented by (biv)below (yield: 80%). Then, the compound was reacted with butyllithium inanhydrous ether to obtain a butyl alcohol form (compound represented by(bv) below) (yield: 73%). Next, the alcohol was converted to a sodiumsalt with sodium hydroxide in anhydrous tetrahydrofuran, and methyliodide was then added thereto, followed by Williamson synthesis toobtain an ether form (compound represented by (bvi) below) (yield: 95%).Next, deprotection was carried out using tetra-n-butylammonium fluoridein anhydrous tetrahydrofuran to obtain an alcohol form (compoundrepresented by (bvii) below) (yield: 87%). Next, the primary hydroxygroup was replaced by chlorine using N-chlorosuccinimide and dimethylsulfide in an anhydrous dichloromethane solvent at −40° C. or lower toobtain a chloride (compound represented by (bviii) below) (yield: 92%).Next, the chloride was diphosphorylated using tris-tetra-n-butylammoniumhydrogen pyrophosphate in anhydrous acetonitrile to obtain a compoundrepresented by (bix) below (compound represented by the formula (B)) asthe substance of interest (yield: 26%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 3 Synthesis of8-hydroxy-3,7-dimethyl-dodeca-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (C))

Synthesis was carried out with geraniol as a starting material. Geraniolwas acetylated using pyridine and acetic anhydride in anhydrousdichloromethane to obtain an acetate (compound represented by (ci)below) (yield: 97%). Next, the carbon atom at position 8 was subjectedto selenium oxidation in ethanol to obtain an aldehyde form (compoundrepresented by (cii) below) (yield: 20%). Next, the aldehyde wasalkaline-hydrolyzed using potassium hydroxide to obtain an alcohol form(compound represented by (ciii) below) (yield: 42%). Next, the alcoholwas treated with imidazole and tert-butyldiphenylsilyl chloride (TBDPS)in anhydrous dichloromethane to obtain a compound represented by (civ)below (yield: 80%). Then, the compound was reacted with butyllithium inanhydrous ether to obtain a butyl alcohol form (yield: 62%). Next,deprotection was carried out using tetra-n-butylammonium fluoride inanhydrous tetrahydrofuran to obtain a diol form (compound represented by(cvi) below) (yield: 94%). Next, the primary hydroxy group was replacedby chlorine using N-chlorosuccinimide and dimethyl sulfide in ananhydrous dichloromethane solvent at −40° C. or lower to obtain achloride (compound represented by (cvii) below) (yield: 90%). Next, thechloride was diphosphorylated using tris-tetra-n-butylammonium hydrogenpyrophosphate in anhydrous acetonitrile to obtain a compound representedby (cviii) below (compound represented by the formula (C)) as thesubstance of interest (yield: 46%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 4 Synthesis of(10S)-hydroxy-3,7-dimethyl-dodeca-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (D))

Synthesis was carried out with farnesol as a starting material. Aprotective group was bonded to the hydroxy group of farnesol usingimidazole, anhydrous dimethylformamide, anhydrous dichloromethane, andtert-butyldiphenylsilyl chloride (TBDPS) to obtain a TBDPS-protectedform (compound represented by (di) below) (yield: 99%). Next, thiscompound was reacted with m-chloroperbenzoic acid in an anhydrousdichloromethane solvent to obtain an epoxy form (compound represented by(dii) (below) (yield: 29%). Next, the epoxy was subjected to periodicacid oxidation using orthoperiodic acid in a mixed solvent of ether andtetrahydrofuran to obtain an aldehyde form (compound represented by(diii) below) (yield: 73%). A Grignard reagent was prepared from ethyliodide and magnesium in an anhydrous ether solvent and then reacted withthe aldehyde to form an alcohol form (compound represented by (div)below) (yield: 80%). The secondary hydroxy group of the racemic alcoholform and (S)-MaNP acid were subjected to reaction in the presence ofN,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and(+)-10-camphorsulfonic acid in an anhydrous dichloromethane solvent toobtain diastereomers. Then, the diastereomers were optically resolved byHPLC, and the absolute configuration was determined by NMR to opticallyobtain a compound represented by (dv) below (yield: 80%). The TBDPSprotective group was then deprotected using tetra-n-butylammoniumhydrate in anhydrous tetrahydrofuran to obtain a diol form (compoundrepresented by (dvi) below) (yield: 80%). Next, the primary hydroxygroup was replaced by chlorine using N-chlorosuccinimide and dimethylsulfide in an anhydrous dichloromethane solvent at −40° C. or lower toobtain a chloride (compound represented by (dvii) below) (yield: 80%).Next, the chloride was diphosphorylated using tris-tetra-n-butylammoniumhydrogen pyrophosphate in anhydrous acetonitrile to obtain a compoundrepresented by (dviii) below (compound represented by the formula (D))as the substance of interest (yield: 46%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 5 Synthesis of(10R)-hydroxy-3,7-dimethyl-dodeca-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (E))

Synthesis was carried out with farnesol as a starting material. Aprotective group was bonded to the hydroxy group of farnesol usingimidazole, anhydrous dimethylformamide, anhydrous dichloromethane, andtert-butyldiphenylsilyl chloride (TBDPS) to obtain a TBDPS-protectedform (compound represented by (ei) below) (yield: 99%). Next, thiscompound was reacted with m-chloroperbenzoic acid in an anhydrousdichloromethane solvent to obtain an epoxy form (compound represented by(eii) below) (yield: 29%). Next, the epoxy was subjected to periodicacid oxidation using orthoperiodic acid in a mixed solvent of ether andtetrahydrofuran to obtain an aldehyde form (compound represented by(eiii) below) (yield: 73%). A Grignard reagent was prepared from ethyliodide and magnesium in an anhydrous ether solvent and then reacted withthe aldehyde to form an alcohol form (compound represented by (eiv)below) (yield: 80%). The secondary hydroxy group of the racemic alcoholform and (S)-MaNP acid were subjected to reaction in the presence ofN,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and(+)-10-camphorsulfonic acid in an anhydrous dichloromethane solvent toobtain diastereomers. Then, the diastereomers were optically resolved byHPLC, and the absolute configuration was determined by NMR to opticallyobtain a compound represented by (ev) below (yield: 84%). The TBDPSprotective group was then deprotected using tetra-n-butylammoniumhydrate in anhydrous tetrahydrofuran to obtain a diol form (compoundrepresented by (evi) below) (yield: 91%). Next, the primary hydroxygroup was replaced by chlorine using N-chlorosuccinimide and dimethylsulfide in an anhydrous dichloromethane solvent at −40° C. or lower toobtain a chloride (compound represented by (evii) below) (yield: 70%).Next, the chloride was diphosphorylated using tris-tetra-n-butylammoniumhydrogen pyrophosphate in anhydrous acetonitrile to obtain a compoundrepresented by (eviii) below (compound represented by the formula (E))as the substance of interest (yield: 59%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 6 Synthesis of10-hydroxy-3,7-dimethyl-dodeca-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (R))

Synthesis was carried out with farnesol as a starting material. Aprotective group was bonded to the hydroxy group of farnesol usingimidazole, anhydrous dimethylformamide, anhydrous dichloromethane, andtert-butyldiphenylsilyl chloride (TBDPS) to obtain a TBDPS-protectedform (fi) (yield: 99%). Next, this compound was reacted withm-chloroperbenzoic acid in an anhydrous dichloromethane solvent toobtain an epoxy form (fii) (yield: 29%). The epoxy was subjected toperiodic acid oxidation using orthoperiodic acid in a mixed solvent ofether and tetrahydrofuran to obtain an aldehyde form (fiii) (yield:73%). A Grignard reagent was prepared from ethyl iodide and magnesium inan anhydrous ether solvent and then reacted with the aldehyde to form analcohol form (fiv) (yield: 80%). The TBDPS group in the compound (fiv)was removed using tetrabutylammonium fluoride in an anhydroustetrahydrofuran solvent to form a diol form (fv) (yield: 93%).

The hydroxy group present at an allylic position in the diol form (fv)was chlorinated using N-chlorosuccinimide and dimethyl sulfide in ananhydrous dichloromethane solvent at −40° C. or lower to form a chloride(fvi) (yield: 69%). The chloride (fvi) was diphosphorylated usingtris(tetra-N-butyl)ammonium hydrogen pyrophosphate in an anhydrousacetonitrile solvent and applied to an ion-exchange column to synthesizea compound (fvii) (compound represented by the formula (R)) as thesubstance of interest (yield: 28%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 7 Synthesis of8-[(tert-butyldimethylsilyl)oxy]-3,7-dimethyl-octa-(2E,6E)-dienyldiphosphate (compound represented by the formula (P))

Synthesis was carried out with geraniol as a starting material. Geraniolwas acetylated using pyridine and acetic anhydride in anhydrousdichloromethane to obtain an acetate (compound represented by (gi)below) (yield: 97%). Next, the carbon atom at position 8 was subjectedto selenium oxidation in ethanol to obtain an alcohol form (compoundrepresented by (gii) below) (yield: 20%). The alcohol was reacted withtert-butyldimethylsilyl chloride in the presence of a basic catalystusing imidazole to obtain a compound represented by (giii) below (yield:87%). Next, the compound was hydrolyzed with potassium hydroxide toobtain an alcohol form (compound represented by (giv) below) (yield:78%). The alcohol form was chlorinated by the N-chlorosuccinimide methodto obtain a chloride (compound represented by (gv) below) (yield: 40%).Next, the chloride was mixed with n-butylammonium hydrogen diphosphatein anhydrous acetonitrile to obtain a compound represented by (gvi)below (compound represented by the formula (P)) as the substance ofinterest (yield: 26%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 8 Synthesis of8-methoxymethoxy-3,7-dimethyl-octa-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (H))

Synthesis was carried out with geraniol as a starting material. Geraniolwas stirred together with acetic anhydride in pyridine to acetylate thehydroxy group, so that a compound represented by (hi) below was obtained(yield: 12%). Next, the compound was subjected to oxidation reactionwith selenium dioxide and tert-butyl hydroperoxide to obtain a transalcohol form (compound represented by (hii) below) (yield: 38%). Thealcohol form was subjected to reaction using chloromethyl ethyl etherand diisopropylethylamine in dichloromethane to obtain a compoundrepresented by (hiii) below in which a methoxymethyl ether group wasintroduced in the hydroxy group at position 8 (yield: 76%). Next, theacetyl group was converted to a hydroxy group using potassium hydroxideto obtain an alcohol form (compound represented by (hiv) below) (yield:95%). The hydroxy group in the alcohol form was chlorinated usingN-chlorosuccinimide to obtain a chloride (compound represented by (hv)below) (yield: 30%). Then, the chloride was diphosphorylated withtris(tetra-n-butyl)ammonium hydrogen diphosphate and applied to acellulose column to obtain a compound represented by (hvi) below(compound represented by the formula (H)) as the substance of interest(yield: 77%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 9 Synthesis of8-n-propylthio-3,7-dimethyl-octa-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (M))

Synthesis was carried out with geraniol as a starting material. Geranioland dihydropyran were condensed in the presence of a pyridiniump-toluenesulfonate catalyst to obtain a compound represented by (ii)below in which the hydroxy group in geraniol was protected with atetrahydropyran ring (yield: 85%). Next, the compound was subjected tooxidation reaction with selenium dioxide and tert-butyl hydroperoxide toobtain a trans alcohol form (compound represented by (iii) below)(yield: 47%). The alcohol form was chlorinated using N-chlorosuccinimidein dichloromethane to obtain a compound represented by (iiii) below(yield: 92%). Next, n-propanethiol was added to a solution of metallicsodium dissolved in ethanol and the resulting solution was reacted withthe compound to obtain a thioether (compound represented by (iiv) below)(yield: 28%). Next, p-toluenesulfonic acid was allowed to act thereon inmethanol to obtain an alcohol form (compound represented by (iv) below)(yield: 75%). The alcohol form was chlorinated by theN-chlorosuccinimide method to obtain a chloride (compound represented by(ivi) below) (yield: 40%). Next, the chloride was mixed withn-butylammonium hydrogen diphosphate in anhydrous acetonitrile to obtaina compound represented by (ivii) below (compound represented by theformula (M)) as the substance of interest (yield: 32%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 10 Synthesis of8-benzyloxy-3,7-dimethyl-octa-(2E,6E)-dienyl diphosphate (compoundrepresented by the formula (N))

Synthesis was carried out with geraniol as a starting material. Geranioland dihydropyran were condensed in the presence of a pyridiniump-toluenesulfonate catalyst to obtain a compound represented by (ji)below in which the hydroxy group in geraniol was protected with atetrahydropyran ring (yield: 85%). Next, the compound was subjected tooxidation reaction with selenium dioxide and tert-butyl hydroperoxide toobtain a trans alcohol form (compound represented by (jii) below)(yield: 47%). Next, thereto were added benzyl bromide and sodiumhydroxide in anhydrous tetrahydrofuran to obtain an ether (compoundrepresented by (jiii) below) (yield: 87%). Next, p-toluenesulfonic acidwas allowed to act thereon in methanol to obtain an alcohol form(compound represented by (jiv) below) (yield: 69%). The alcohol form waschlorinated by the N-chlorosuccinimide method to obtain a chloride(compound represented by (jv) below) (yield: 40%). Next, the chloridewas mixed with n-butylammonium hydrogen diphosphate in anhydrousacetonitrile to obtain a compound represented by (jvi) below (compoundrepresented by the formula (N)) as the substance of interest (yield:26%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 11 Synthesis of7-acetyl-7-aza-3-methyl-dodeca-(2E)-dienyl diphosphate (compoundrepresented by the formula (K))

Synthesis was carried out with geraniol as a starting material. Geraniolwas acetylated with acetic anhydride in the presence ofdimethylaminopyridine in anhydrous dichloromethane to obtain an esterform (compound represented by (ki) below) (yield: 96%). The olefin atposition 6 was oxidized using m-chloroperbenzoic acid in anhydrousdichloromethane to obtain an epoxy form (compound represented by (kii)below) (yield: 92%). Next, the epoxy was oxidized with orthoperiodicacid in anhydrous tetrahydrofuran to obtain an aldehyde form (compoundrepresented by (kiii) below) (yield: 66%). Next, the aldehyde wasreductively aminated using n-butylamine and sodium cyanoborohydride inanhydrous methanol to obtain a secondary amine (compound represented by(kiv) below) (yield: 66%). The amine was acetylated with aceticanhydride in the presence of dimethylaminopyridine in an anhydrousdichloromethane solvent to obtain a secondary amide (compoundrepresented by (kv) below) (yield: 38%). The ester site was irreversiblyhydrolyzed with potassium hydroxide in anhydrous methanol to obtain aprimary alcohol form (compound represented by (kvi) below) (yield: 76%).Next, the primary hydroxy group was replaced by chlorine usingN-chlorosuccinimide and dimethyl sulfide in an anhydrous dichloromethanesolvent at −40° C. or lower to obtain a chloride (compound representedby (kvii) below) (yield: 67%). Next, the chloride was diphosphorylatedusing tris-tetra-n-butylammonium hydrogen pyrophosphate in anhydrousacetonitrile to obtain the substance of interest (compound representedby (kviii) below (compound represented by the formula (K))) (yield:50%).

The intermediate in each synthesis step and the final product wereconfirmed by TLC and instrumental analysis (IR, NMR).

Production Example 12 Preparation of Mutagenized Enzyme

The reagent used was QuikChange Site-Directed Mutagenesis Kit fromStratagene Corp. Primers were designed so that they allowed mutagenesisat a site of interest. Primers for mutagenesis were purchased fromMedical & Biological Laboratories, Co., Ltd. (manufacturer: XX IDT). Thedesigned primers are as follows.

Primers for preparation of variant enzyme N31A:

Sense primer 5′-gac gga gca ggc cga tgg gca aaa-3′ (SEQ ID NO: 23),

Antisense primer 5′-cat cgg cct gct ccg tcc ata atg a-3′ (SEQ ID NO:24);

Primers for preparation of variant enzyme N77A:

Sense primer 5′-act gaa gca tgg tct cgt cct aaa g-3′ (SEQ ID NO: 25),

Antisense primer 5′-gag acc atg ctt cag ttg aaa atg c-3′ (SEQ ID NO:26);

Primers for preparation of variant enzyme L91N:

Sense primer 5′-gat gaa aaa ccc ggg tga ttt ttt aa-3′ (SEQ ID NO: 27),

Antisense primer 5′-cac ccg ggt ttt tca tca agt aat ta-3′ (SEQ ID NO:28);

Primers for preparation of variant enzyme L91D:

Sense primer 5′-gat gaa aga tcc ggg tga ttt ttt aa-3′ (SEQ ID NO: 29),

Antisense primer 5′-cac ccg gat ctt tca tca agt aat ta-3′ (SEQ ID NO:30);

Primers for preparation of variant enzyme N31Q:

Sense primer 5′-gac gga caa ggc cga tgg gca aaa-3′ (SEQ ID NO: 31),

Antisense primer 5′-cca tcg gcc ttg tcc gtc cat aat-3′ (SEQ ID NO: 32);

Primers for preparation of variant enzyme N77Q:

Sense primer 5′-act gaa caa tgg tct cgt cct aaa g-3′ (SEQ ID NO: 33),

Antisense primer 5′-cga gac cat gct tca gtt gaa aat gc-3′ (SEQ ID NO:34);

Primers for preparation of variant enzyme L91G:

Sense primer 5′-gat gaa agg acc ggg tga ttt ttt aa-3′ (SEQ ID NO: 35),

Antisense primer 5′-acc cgg tcc ttt cat caa gta att aac-3′ (SEQ ID NO:36);

Primers for preparation of variant enzyme L91K:

Sense primer 5′-gat gaa aaa acc ggg tga ttt ttt aa-3′ (SEQ ID NO: 37),

Antisense primer 5′-acc cgg ttt ttt cat caa gta att aa-3′ (SEQ ID NO:38);

Primers for preparation of variant enzyme F95A:

Sense primer 5′-ggg tga tgc gtt aaa cac att ttt ac-3′ (SEQ ID NO: 39),

Antisense primer 5′-gtt taa tgc atc acc cgg tag ttt ca-3′ (SEQ ID NO:40);

Primers for preparation of variant enzyme F95W:

Sense primer 5′-ggg tga ttg gtt aaa cac att ttt ac-3′ (SEQ ID NO: 41),

Antisense primer 5′-gtt taa cca atc acc cgg tag ttt ca-3′ (SEQ ID NO:42).

The dsDNA template used was pET22b containing the base sequence ofMicrococcus luteus B-P 26-derived undecaprenyl diphosphate synthase(hereinafter, also referred to as the wild-type enzyme) (this templateis referred to as pET22b/MLU-UPS). This pET22b/MLU-UPS was kindlyprovided by Professor Tanetoshi Koyama (Institute of MultidisciplinaryResearch for Advanced Materials Tohoku University). 2 μl of 10×Pfupolymerase buffer was mixed with 2-20 ng of the dsDNA template, 50 ng ofthe sense primer, 50 ng of the antisense primer, 0.4 μl of dNTPs (2.5 mMeach), ddH₂O up to 20 μl, 0.4 ml of Pfu polymerase (2.5 U/μl) and PCRreaction was then performed. The PCR reaction was performed under thefollowing conditions: 1 cycle of 95° C. for 30 sec; 15 cycles of 95° C.for 30 sec, 55° C. for 1 min, 68° C. for 8 min. After PCR, 0.4 μl ofDpnI was added to the PCR reaction solution and DpnI treatment wasperformed at 37° C. for 1 hour. E. coli DH5α was transformed by the heatshock method using 1-10 μl of the DpnI-treated solution, and thetransformant was applied to an LB agar medium containing 50 μg/mLampicillin and then cultured overnight at 37° C., and then thetransformed strain was selected. The transformant was cultured all nightin an LB medium containing 50 μg/ml ampicillin. A plasmid was preparedby the alkali-SDS method from the obtained culture solution. Mutagenesisin the plasmid was confirmed using a sequencer.

Production Example 13 Production of Protein Having PrenyltransferaseActivity

E. coli BL21 (DE3) was transformed with each plasmid pET22b/MLU-UPS(wild-type and variant). The obtained E. coli BL21(DE3)/pET22b/MLU-UPS(wild-type and variant) was inoculated into a test tube containing 3 mLof an LB medium containing 50 μg/mL ampicillin and shake-cultured at 37°C. for 5 hours. A 1 mL aliquot of the obtained culture solution wasinoculated into a 500-mL Erlenmeyer flask containing 100 mL of an LBmedium containing 50 μg/mL ampicillin and shake-cultured at 37° C. for 3hours. Then, IPTG was added thereto at a concentration of 0.1 mmol/L,and the bacterial cells were shake-cultured at 30° C. for 18 hours. Theculture solution was centrifuged to obtain wet bacterial cells.

The wet bacterial cells thus obtained were disrupted by sonication andthen centrifuged. A protein having prenyltransferase activity waspurified from the obtained supernatant using HisTrap (manufactured byAmersham Biosciences Corp.). The purification of the purified proteinwas confirmed by SDS-PAGE.

Examples and Comparative Examples Preparation of Isoprene Oligomer

A reaction solution containing 10 mg of each purified protein, 50 mMTris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100,25 mM 2-mercaptoethanol, 1 mM of an initiating substrate (farnesyldiphosphate or each of the initiating substrates prepared in ProductionExamples 1 to 11), and 1 mM isopentenyl diphosphate was prepared andreacted for 1 hour in a water bath at 37° C.

After the completion of reaction, 100 ml of saturated saline and 500 mlof 1-butanol were added thereto, and the mixture was stirred and thenleft at rest. Then, the supernatant (1-butanol layer) was concentratedto dryness by evaporation. A portion of the residue was structurallyconfirmed by NMR to obtain an isoprene oligomer.

The details (n and m in the formula (1)) of the thus obtained isopreneoligomers are as shown in Tables 1 and 2. Here, Y was a hydroxy group ora group represented by the formula (2).

In these examples, n and m in the formula (1) were calculated on thebasis of information about the initiating substrate used and the lengthof the isoprene chain determined by TLC. Also, Y was structurallyidentified by NMR or IR.

TABLE 1 Initiating substrate Compound Compound Compound CompoundCompound Compound Farnesyl of formula of formula of formula of formulaof formula of formula n diphosphate (S) (B) (C) (D) (E) (R) Wild-typeenzyme 3 2 2 2 2 2 2 Variant enzyme N31A 3 2 2 2 2 2 2 N77A 3 2 2 2 2 22 L91N 3 2 2 2 2 2 2 L91D 3 2 2 2 2 2 2 N31Q 3 2 2 2 2 2 2 N77Q 3 2 2 22 2 2 L91G 3 2 2 2 2 2 2 L91K 3 2 2 2 2 2 2 F95A 3 2 2 2 2 2 2 F95W 3 22 2 2 2 2 Initiating substrate Compound Compound Compound CompoundCompound of formula of formula of formula of formula of formula n (P)(H) (M) (N) (K) Wild-type enzyme 2 2 2 2 1 Variant enzyme N31A 2 2 2 2 1N77A 2 2 2 2 1 L91N 2 2 2 2 1 L91D 2 2 2 2 1 N31Q 2 2 2 2 1 N77Q 2 2 2 21 L91G 2 2 2 2 1 L91K 2 2 2 2 1 F95A 2 2 2 2 1 F95W 2 2 2 2 1

TABLE 2 Initiating substrate Compound Compound Compound CompoundCompound Compound Farnesyl of formula of formula of formula of formulaof formula of formula m diphosphate (S) (B) (C) (D) (E) (R) Wild-typeenzyme 7, 8, 9 3, 4, 5, 6, 7 5, 6, 7, 8, 9 4, 5, 6, 7 4, 5, 6, 7 4, 5,6, 7 4, 5, 6, 7 Variant enzyme N31A 7, 8, 9 4, 5, 6, 7 4, 5, 6, 7, 8, 94, 5, 6, 7 4, 5, 6, 7 4, 5, 6, 7 4, 5, 6, 7 N77A 8, 9 1, 4, 5, 6 6, 7,8, 1, 4, 5, 6 5, 6 5, 6 5, 6 L91N 8, 9 3, 4, 5 6, 7, 8, 3, 4, 5, 6 3, 4,5 3, 4, 5 3, 4, 5 L91D 8, 9 3, 4, 5 5, 6, 7, 8 4, 5, 6 4, 5, 6 4, 5 4,5, 6 N31Q 7, 8, 9 4, 5, 6, 7 4, 5, 6, 7, 8, 9 4, 5, 6, 7 4, 5, 6, 7 4, 54, 5, 6, 7 N77Q 8, 9 3, 4, 5 4, 5, 6, 7, 8, 9 4, 5, 6, 7, 8 4, 5, 6 4, 54, 5, 6, 7 L91G 7, 8, 9 3, 4, 5 6, 7, 8, 3, 4, 5, 6 3, 4, 5 4, 5, 6, 74, 5, 6, 7 L91K 8, 9 3, 4, 5 6, 7, 8, 3, 4, 5, 6 4, 5, 6, 7 4, 5 4, 5,6, 7 F95A 8, 9, 10 3, 4, 5 5, 6, 7, 8 3, 4, 5, 6, 7 5, 6, 7, 8 4, 5, 6,7, 8 3, 4, 5 F95W 8, 9, 10 3, 4, 5 5, 6, 7, 8 3, 4, 5, 6, 7 5, 6, 7, 84, 5, 6, 7, 8 3, 4, 5 Initiating substrate Compound Compound CompoundCompound Compound of formula of formula of formula of formula of formulam (P) (H) (M) (N) (K) Wild-type enzyme 3, 4, 5, 6 7, 8, 9 4, 5, 6, 7 3,4, 5, 6 3, 4, 5, 6 Variant enzyme N31A 3, 4, 5, 6 7, 8, 9 4, 5, 6 4, 5,6, 7 5, 6, 7 N77A 2, 3, 4, 5, 6 7, 8, 9 4, 5, 6 4, 5, 6 4, 5, 6 L91N 2,3, 4, 5, 6 8, 9 3, 4, 5, 6 3, 4, 5 3, 4, 5 L91D 3, 4, 5, 6 8, 9 4, 5, 63, 4, 5 3, 4, 5 N31Q 2, 3, 4, 5, 6 7, 8, 9 4, 5, 6, 7 5, 6, 7 5, 6, 7N77Q 2, 3, 4, 5, 6 8, 9 4, 5, 6 3, 4, 5 3, 4, 5 L91G 4, 5, 6 7, 8, 9 3,4, 5, 6 3, 4, 5, 6 4, 5, 6 L91K 4, 5 8, 9 3, 4, 5, 6 3, 4, 5 3, 4, 5F95A 4, 5 7, 8, 9 3, 4, 5, 6, 7 3, 4, 5 3, 4, 5, 6 F95W 4, 5 7, 8, 9 3,4, 5, 6, 7 3, 4, 5 3, 4, 5, 6

Examples and Comparative Examples Comparison of Activity BetweenWild-Type Enzyme and Variant Enzyme (Initiating Substrate-Based RelativeActivity)

Reaction was performed under conditions shown below using each of theinitiating substrates prepared in Production Examples 1 to 11 orfarnesyl diphosphate. The activity of each variant enzyme on eachinitiating substrate was indicated by index with the activity of thewild-type enzyme (Micrococcus luteus B-P 26-derived undecaprenyldiphosphate synthase) as 100.

A reaction solution containing 500 ng of each purified protein, 50 mMTris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100,25 mM 2-mercaptoethanol, 12.5 μM of an initiating substrate, and 50 μM[1-¹⁴C] isopentenyl diphosphate was prepared and reacted for 1 hour in awater bath at 37° C. After the reaction, liquid scintillation countingand TLC quantification were performed to measure the activity of eachenzyme.

TABLE 3 Initiating substrate Compound Compound Compound CompoundCompound Compound Farnesyl of formula of formula of formula of formulaof formula of formula Relative activity diphosphate (S) (B) (C) (D) (E)(R) Wild-type enzyme 100 100 100 100 100 100 100 Variant enzyme N31A 123116 116 111 179 222 200 N77A 213 — 319 234 208 396 254 L91N 156 — — 108103 271 137 L91D 231 227 231 155 125 320 230 N31Q — — — — 104 210 146N77Q 121 — — — 124 194 156 L91G — 130 149 146 134 170 110 L91K 133 147156 130 101 163 165 F95A — 163 128 — — 120 140 F95W — 130 140 — 120 114121 Initiating substrate Compound Compound Compound Compound Compound offormula of formula of formula of formula of formula Relative activity(P) (H) (M) (N) (K) Wild-type enzyme 100 100 100 100 100 Variant enzymeN31A — 152 149 — — N77A — 162 127 112 — L91N — — 139 — — L91D 112 — 123— — N31Q — 110 101 — — N77Q — — — 133 — L91G 104 — — — — L91K — — 112 —112 F95A 104 — — 132 136 F95W 130 — — 147 148

Examples and Comparative Examples Preparation of Polyisoprene

A reaction solution containing 10 μl of a latex component, 50 mMTris-HCl buffer (pH 7.5), 25 mM magnesium chloride, 40 mM2-mercaptoethanol, 40 mM potassium fluoride, 50 μM of an isopreneoligomer, and 1 mM isopentenyl diphosphate was prepared and reacted for3 days in a water bath at 30° C. After the reaction, the molecularweight was measured by GPC. Then, n and q in the formula (4) werecalculated on the basis of the measured molecular weight and informationabout the initiating substrate used. The details (n and q in the formula(4)) of the thus obtained polyisoprenes are as shown in Tables 4 and 5.Here, Y was a hydroxy group or a group represented by the formula (2).Also, Y was identified in the same way as in Examples (Preparation ofisoprene oligomer).

The latex component used was a serum prepared by the ultracentrifugationof latex taken from Hevea brasiliensis.

Each isoprene oligomer used was an isoprene oligomer obtained using thevariant enzyme N31A or the like and each initiating substrate (farnesyldiphosphate or each of the initiating substrates prepared in ProductionExamples 1 to 11) under the same conditions as in Examples (Preparationof isoprene oligomer). In the description below, the isoprene oligomersobtained using the variant enzyme N31A or the like together withfarnesyl diphosphate, the compound represented by the formula (S), thecompound represented by the formula (B), the compound represented by theformula (C), the compound represented by the formula (D), the compoundrepresented by the formula (E), the compound represented by the formula(R), the compound represented by the formula (P), the compoundrepresented by the formula (H), the compound represented by the formula(M), the compound represented by the formula (N), and the compoundrepresented by the formula (K) as initiating substrates under the sameconditions as in Examples (Preparation of isoprene oligomer) arereferred to as isoprene oligomer (0), isoprene oligomer (S), isopreneoligomer (B), isoprene oligomer (C), isoprene oligomer (D), isopreneoligomer (E), isoprene oligomer (R), isoprene oligomer (P), isopreneoligomer (H), isoprene oligomer (M), isoprene oligomer (N), and isopreneoligomer (K), respectively.

TABLE 4 Isoprene oligomer Isoprene Isoprene Isoprene Isoprene IsopreneIsoprene Isoprene oligomer oligomer oligomer oligomer oligomer oligomeroligomer n (O) (S) (B) (C) (D) (E) (R) Wild-type enzyme 3 2 2 2 2 2 2Variant enzyme N31A 3 2 2 2 2 2 2 N77A 3 2 2 2 2 2 2 L91N 3 2 2 2 2 2 2L91D 3 2 2 2 2 2 2 N31Q 3 2 2 2 2 2 2 N77Q 3 2 2 2 2 2 2 L91G 3 2 2 2 22 2 L91K 3 2 2 2 2 2 2 F95A 3 2 2 2 2 2 2 F95W 3 2 2 2 2 2 2 Isopreneoligomer Isoprene Isoprene Isoprene Isoprene Isoprene oligomer oligomeroligomer oligomer oligomer n (P) (H) (M) (N) (K) Wild-type enzyme 2 2 22 1 Variant enzyme N31A 2 2 2 2 1 N77A 2 2 2 2 1 L91N 2 2 2 2 1 L91D 2 22 2 1 N31Q 2 2 2 2 1 N77Q 2 2 2 2 1 L91G 2 2 2 2 1 L91K 2 2 2 2 1 F95A 22 2 2 1 F95W 2 2 2 2 1

TABLE 5 Isoprene oligomer Isoprene Isoprene Isoprene Isoprene IsopreneIsoprene Isoprene oligomer oligomer oligomer oligomer oligomer oligomeroligomer q (O) (S) (B) (C) (D) (E) (R) Wild-type enzyme 5000-30000 3000-10000 3000-15000 5000-30000 4500-15000 3000-12000  3000-12000Variant enzyme N31A 3000-15000 1000-7350 3000-15000 3000-100003000-10000 3000-12000  3000-12000 N77A 3000-10000 1000-7350 4000-120003000-10000 5000-20000 5000-10000  5000-10000 L91N 1000-12000 1000-65004000-12000 1000-12000 5000-20000 5000-8000  5000-8000 L91D 10000-30000 1000-6500 3000-10000 1000-12000 1000-12000 1500-7000  1500-7000 N31Q3000-15000  2000-10000 3000-15000 3500-30000 1000-12000 2000-7500 2000-7500 N77Q 3000-15000 1000-7350 3000-15000 3000-15000 3500-100001500-6500  1500-6500 L91G 3000-15000 1000-7350 3000-10000 3000-200003000-15000 1500-7500  2000-7500 L91K 3000-15000 1500-7350 3000-100003000-15000 1500-8000  3000-12000 1500-6500 F95A 700-1000 3000-73505000-20000 2000-10000 1700-7500  1500-10000 1500-8000 F95W 700-10003000-7350 5000-20000 1500-8000  1500-7500  1500-10000 1000-8000 Isopreneoligomer Isoprene Isoprene Isoprene Isoprene Isoprene oligomer oligomeroligomer oligomer oligomer q (P) (H) (M) (N) (K) Wild-type enzyme1000-3500  1000-12000  4000-10000 1500-7000 100-1500 Variant enzyme N31A 800-5000  3000-12000  3000-10000 1500-5000 150-1500 N77A  800-50001500-7000 2000-7000 3000-6000 200-1500 L91N 1000-2000 2000-75002000-7500 3000-6000 200-1500 L91D 1000-3000 1500-6500 1500-65001000-7500 150-2500 N31Q 1500-4000 1500-7000 1000-7000 1500-6000 100-1500N77Q 1000-3000 2000-7500 2000-7500 2000-5000 250-2000 L91G 1000-25001500-6500  1500-10000 1500-5000 250-1500 L91K 1000-4500 2000-75002000-7500 2500-8000 100-1500 F95A  450-1500 2000-7000 2000-65003000-8000 250-2500 F95W  450-2000 1500-6500 1500-6500 3000-8000 250-2500(Antimicrobial Test)

An antimicrobial test was conducted using isoprene oligomers obtainedusing the variant enzyme N31A under the same conditions as in Examples(Preparation of isoprene oligomer). The isoprene oligomers used were theisoprene oligomer (0), the isoprene oligomer (S), the isoprene oligomer(B), the isoprene oligomer (C), the isoprene oligomer (D), the isopreneoligomer (E), the isoprene oligomer (R), the isoprene oligomer (P), theisoprene oligomer (H), the isoprene oligomer (M), the isoprene oligomer(N), and the isoprene oligomer (K).

The following microbial strains were used in the antimicrobial test:

Gram-positive bacteria: Staphylococcus aureus (13276) and Bacillussubtilis (3134)

Gram-negative bacteria: Echerichia coli (3972), Salmonella enteric(100797), Peudomonas aeruginosa (13275), and Klebsiella pneumonia (3512)

Fungi: Candida albicans (1594)

The number in the parentheses represents NBRC No. from NationalInstitute of Technology and Evaluation, Biological Resource Center. Allthe microbial strains used were purchased from NBRC.

Next, the recovery and culture conditions of the microbial strains usedare shown in Table 6.

TABLE 6 Culture NBRC Liquid Solid temperature No. culture medium (° C.)1594 703 108 24 3134 702 802 30 3512 702 802 30 3972 702 802 30 13275702 802 30 13276 702 802 30 100797 702 802 30Composition of each medium (1 L solution each)108: 10 g of glucose, 5 g of peptone, 3 g of yeast extract, 3 g of maltextract, and 15 g of agar, pH 5.6;702: 10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO₄.7H₂O,pH 7.0;703: 10 g of glucose, 5 g of peptone, 3 g of yeast extract, and 3 g ofmalt extract, pH 6.0;802: 10 g of polypeptone, 2 g of yeast extract, 1 g of MgSO₄.7H₂O, and15 g of agar, pH 7.0.

The Staphylococcus aureus strain was inoculated into a test tubecontaining 2 ml of a liquid medium (10 g/L polypeptone, 2 g/L yeastextract, and 1 g/L MgSO₄.7H₂O, pH 7.0) and cultured at 150 rpm at 30° C.for 5 hours. The microbial strain was inoculated into a solid medium (10g/L polypeptone, 2 g of yeast extract, 1 g/L MgSO₄.7H₂O, and 15 g/Lagar, pH 7.0) and cultured overnight at 30° C. On the next day, colonieswere confirmed. One of the colonies on the solid medium was inoculatedinto a test tube containing 4 ml of the liquid medium and cultured(i.e., precultured) overnight at 30° C. Next, 100 μl of the preculturesolution was added to a new test tube containing 4 ml of the liquidmedium and cultured at 150 rpm at 30° C. During the culture, theturbidity was measured using a spectrophotometer, and the culture wascontinued until O.D.600=0.1 to 0.3. The culture solution was adjusted to10⁵ cfu/ml by the dilution method and used in the antimicrobial test.

The minimum inhibitory concentration (MIC) was measured by the MICmethod, which is a general experimental approach for examining theactivity of anti-microorganism substances. The microbial culturesolution thus adjusted to 10⁵ cfu/ml and each isoprene oligomer adjustedto 0.5-5 mM were added into a 1.5-ml tube, mixed with each other, andcultured overnight at 150 rpm at 30° C. After the culture, eachconcentration of the culture solution was applied to the solid mediumusing a spreader and cultured at 30° C. for 1-4 days. After the culture,the presence or absence of growth of the microbe was examined by visualobservation, and the minimum addition concentration of the terminallymodified isoprene oligomer at which the microbe did not survive wasdefined as MIC.

MIC was determined for the other microbes in the same way as above.

The isoprene oligomers used and their MICs are shown in Table 7.

TABLE 7 Isoprene oligomer Comparative Example 1 Example 1 Example 2Example 3 Example 4 Example 5 Example 6 Isoprene Isoprene IsopreneIsoprene Isoprene Isoprene Isoprene oligomer oligomer oligomer oligomeroligomer oligomer oligomer MIC(μg/ml) (O) (S) (B) (C) (D) (E) (R)Staphylococcus aureus 13276 * — 260 160 560 460 58 Bacillus subtilis3134 * — 260 160 560 320 58 Echerichia coli 3972 * — 180 — 240 — 130Salmonella enteric 100797 * — — — — — — Peudomonas aeruginosa 13275 * 93— — — — — Klebsiella pneumonia 3512 * 93  58 — — — — Candida albicans1594 * 93 — — 240 90 130 Isoprene oligomer Example 7 Example 8 Example 9Example 10 Example 11 Isoprene Isoprene Isoprene Isoprene Isopreneoligomer oligomer oligomer oligomer oligomer MIC(μg/ml) (P) (H) (M) (N)(K) Staphylococcus aureus 13276 140 — 190 560 45 Bacillus subtilis 3134140 — 760 560 30 Echerichia coli 3972 — — 420 240 30 Salmonella enteric100797 — — 240 37 50 Peudomonas aeruginosa 13275 — — — 37 56 Klebsiellapneumonia 3512 — — — 130 75 Candida albicans 1594 — 88 — 20 35 *Antimicrobial activity was not observed at 800 ppm or lower.(Discussion about Results of Table 7)

The isoprene oligomer (0) of Comparative Example (isoprene oligomerobtained from farnesyl diphosphate) in which at least one atom or groupcontained in the trans structural moiety was not replaced by anotheratom or group did not exhibit antimicrobial activity (antimicrobialactivity was not observed at 800 ppm or lower). In contrast, theisoprene oligomers of Examples in which at least one atom or groupcontained in the trans structural moiety was replaced by another atom orgroup exhibited antimicrobial activity.

(Rubber Composition)

Next, a rubber composition containing the isoprene oligomer and/or thepolyisoprene of the present invention was evaluated for its performance.First, the compound represented by the formula (F) was synthesized inthe same way as in Production Examples 1 to 11.

Next, farnesyl diphosphate and the synthesized initiating substrates(the compound represented by the formula (K) and the compoundrepresented by the formula (F)) were used to prepare isoprene oligomers.The details (n, m, and Y in the formula (1)) of the obtained isopreneoligomers were determined in the same way as in Examples (Preparation ofisoprene oligomer).

Production Example 14 Preparation of Isoprene Oligomer (F)

The isoprene oligomer (F) was prepared from the compound represented bythe formula (F) as an initiating substrate. This reaction was performedby adjusting the conditions described above in (Preparation of isopreneoligomer) in order to adjust the molecular weight of the isopreneoligomer to be obtained.

The details (n and m in the formula (1)) of the obtained isopreneoligomer (F) were n=2 and m=5 to 8. Here, Y was a hydroxy group or agroup represented by the formula (2).

Production Example 15 Preparation of Isoprene Oligomer (K)

The isoprene oligomer (K) was prepared from the compound represented bythe formula (K) as an initiating substrate. This reaction was performedby adjusting the conditions described above in (Preparation of isopreneoligomer) in order to adjust the molecular weight of the isopreneoligomer to be obtained.

The details (n and m in the formula (1)) of the obtained isopreneoligomer (K) were n=1 and m=5 to 7. Here, Y was a hydroxy group or agroup represented by the formula (2).

Production Example 16 Preparation of Isoprene Oligomer (0)

The isoprene oligomer (0) was prepared from farnesyl diphosphate as aninitiating substrate. This reaction was performed by adjusting theconditions described above in (Preparation of isoprene oligomer) inorder to adjust the molecular weight of the isoprene oligomer to beobtained.

The details (n and m in the formula (1)) of the obtained isopreneoligomer (0) were n=3 and m=5 to 7. Here, Y was a hydroxy group or agroup represented by the formula (2).

Next, polyisoprenes were prepared from the isoprene oligomers obtainedin Production Examples 14 to 16 (the isoprene oligomer (F), the isopreneoligomer (K), and the isoprene oligomer (0)). The details (n, q, and Yin the formula (4)) of the obtained polyisoprenes were determined in thesame way as in Examples (Preparation of polyisoprene).

Production Example 17 Preparation of Polyisoprene (F)

The polyisoprene (F) was prepared from the isoprene oligomer (F)prepared in Production Example 14. This reaction was performed byadjusting the conditions described above in (Preparation ofpolyisoprene) in order to adjust the molecular weight of thepolyisoprene to be obtained.

The details (n and q in the formula (4)) of the obtained polyisoprene(F) were n=2 and q=7000 to 12000. Here, Y was a hydroxy group or a grouprepresented by the formula (2).

Production Example 18 Preparation of Polyisoprene (K-1)

The polyisoprene (K-1) was prepared from the isoprene oligomer (K)prepared in Production Example 15. This reaction was performed byadjusting the conditions described above in (Preparation ofpolyisoprene) in order to adjust the molecular weight of thepolyisoprene to be obtained.

The details (n and q in the formula (4)) of the obtained polyisoprene(K-1) were n=1 and q=3500 to 7000. Here, Y was a hydroxy group or agroup represented by the formula (2).

Production Example 19 Preparation of Polyisoprene (K-2)

The polyisoprene (K-2) was prepared from the isoprene oligomer (K)prepared in Production Example 15. This reaction was performed byadjusting the conditions described above in (Preparation ofpolyisoprene) in order to adjust the molecular weight of thepolyisoprene to be obtained.

The details (n and q in the formula (4)) of the obtained polyisoprene(K-2) were n=1 and q=7000 to 12000. Here, Y was a hydroxy group or agroup represented by the formula (2).

Production Example 20 Preparation of Polyisoprene (0)

The polyisoprene (0) was prepared from the isoprene oligomer (0)prepared in Production Example 16. This reaction was performed byadjusting the conditions described above in (Preparation ofpolyisoprene) in order to adjust the molecular weight of thepolyisoprene to be obtained.

The details (n and q in the formula (4)) of the obtained polyisoprene(0) were n=3 and q=7000 to 12000. Here, Y was a hydroxy group or a grouprepresented by the formula (2).

Hereinafter, various agents used in Examples 12 to 21 and ComparativeExamples 2 to 6 will be summarized.

NR: TSR20

BR: BR01 manufactured by JSR Corp.

Carbon black: Diablack (N220) manufactured by Mitsubishi Chemical Corp.

Isoprene oligomer (F), isoprene oligomer (K), and isoprene oligomer (0):isoprene oligomers obtained in Production Examples 14 to 16

Polyisoprene (F), polyisoprene (K-1), polyisoprene (K-2), andpolyisoprene (0): polyisoprenes obtained in Production Examples 17 to 20

Zinc oxide: zinc oxide No. 1 manufactured by Mitsui Mining & SmeltingCo., Ltd.

Stearic acid: stearic acid manufactured by NOF Corp.

Antioxidant: Nocrac 6C(N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine) manufactured byOuchi Shinko Chemical Industrial Co., Ltd.

Wax: Sannoc wax manufactured by Ouchi Shinko Chemical Industrial Co.,Ltd.

Sulfur: sulfur powder manufactured by Tsurumi Chemical Industry Co.,Ltd.

Vulcanization accelerator NS: Nocceler NS(N-tert-butyl-2-benzothiazolesulfenamide) manufactured by Ouchi ShinkoChemical Industrial Co., Ltd.

Silica: Nipsil AQ (wet silica) manufactured by Nippon

Silica Industrial Co., Ltd.

Silane coupling agent: Si266 (bis(3-triethoxysilylpropyl) disulfide)manufactured by Degussa

Vulcanization accelerator DPG: Nocceler D (N,N-diphenylguanidine)manufactured by Ouchi Shinko Chemical Industrial Co., Ltd.

Examples 12 to 21 and Comparative Examples 2 to 6

The materials other than sulfur and vulcanization accelerators werekneaded using a 1.7-L Banbury mixer according to the formulation shownin Table 8 or 9 to obtain a kneaded mixture. Next, sulfur andvulcanization accelerator(s) were added to the obtained kneaded mixtureand kneaded thereinto using an open roll mill to obtain an unvulcanizedrubber composition. The obtained unvulcanized rubber composition wasvulcanized at a pressure of 80 kgf/cm² at 150° C. for 30 minutes using asteam vulcanization press to obtain a vulcanized rubber composition.

The thus obtained vulcanized rubber compositions were evaluated as shownbelow. The results are shown in Tables 8 and 9. The formulations ofComparative Examples 4 and 5 were used as reference formulations inTables 8 and 9, respectively.

(Viscoelasticity Test)

The tan δ was measured under conditions of 70° C. and 2% strain (initialelongation) using a viscoelasticity spectrometer manufactured by IwamotoSeisakusho Co., Ltd. and indicated by index with the tan δ of thereference formulation as 100. A larger index represents larger heatbuild-up. A rubber composition having an index of 100 or less wasregarded as having improved in resistance to heat build-up(low-heat-build-up properties). This means that a rubber compositionhaving a smaller index is more excellent in low-heat-build-upproperties.

(Lambourn Abrasion Test)

An abrasion test was carried out for 5 minutes under conditions of 3 kgload, 40% slip ratio, and sand falling at a rate of 15 g/min using aLambourn abrasion tester manufactured by Iwamoto Seisakusho Co., Ltd.The shape of each sample was set to a thickness of 5 mm and a diameterof 50 mm, and the grindstone used was GC-type abrasive grains having aparticle size of #80. The test results were converted to an index withthe value of the reference formulation as 100 (reference). A largerindex represents more excellent abrasion resistance. A rubbercomposition having an index exceeding 100 was regarded as havingimproved in abrasion resistance.

(Tensile Test)

A tensile test was carried out according to JIS K6251 “Rubber,vulcanized or thermoplastic—Determination of tensile stress-strainproperties” using No. 3 dumbbell test pieces from the vulcanized rubbersheet, and the tensile strength at break (TB) (MPa) and elongation atbreak (EB) (%) were measured. Elongation at break less than 480% tendsto cause rubber separation during use in large tires and thus requiresimprovement. Also as for tensile strength at break, its reduction isresponsible for the destruction of tires and thus it is necessary toprevent a reduction due to the change in materials.

TABLE 8 Comparative Comparative Comparative Example 12 Example 13Example 14 Example 15 Example 2 Example 3 Example 4 Formulation NR 80 8080 80 80 80 80 (Part(s) by mass) BR 20 20 20 20 20 20 20 Carbon black 5050 50 50 50 50 50 Isoprene oligomer (F) 3 10 — — — — — Isoprene oligomer(K) — — 3 10 — — — Isoprene oligomer (O) — — — — 3 10 — Zinc oxide 3 3 33 3 3 3 Stearic acid 2 2 2 2 2 2 2 Antioxidant 2 2 2 2 2 2 2 Wax 1.5 1.51.5 1.5 1.5 1.5 1.5 Sulfur 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Vulcanizationaccelerator NS 1 1 1 1 1 1 1 Performance Viscoelasticity tan δ (index)97 94 96 93 102 104 100 evaluation Lambourn abrasion (index) 104 106 103106 98 96 100 Elongation at break (%) 520 570 520 580 530 570 510

TABLE 9 Comparative Comparative Example 16 Example 17 Example 18 Example19 Example 20 Example 21 Example 5 Example 6 Formulation Polyisoprene(F) 100 — — 80 60 — — — (Part(s) by Polyisoprene (K-1) — 100 — — — 60 —— mass) Polyisoprene (K-2) — — 100 — — — — — Polyisoprene (O) — — — — —— 60 — NR — — — 20 40 40 40 100 Silica 50 50 50 50 50 50 50 50 Silanecoupling 4 4 4 4 4 4 4 4 agent Stearic acid 2 2 2 2 2 2 2 2 Antioxidant2 2 2 2 2 2 2 2 Zinc oxide 3 3 3 3 3 3 3 3 Sulfur 1.5 1.5 1.5 1.5 1.51.5 1.5 1.5 Vulcanization 1 1 1 1 1 1 1 1 accelerator NS Vulcanization 11 1 1 1 1 1 1 accelerator DPG Performance Viscoelasticity 89 93 88 91 9493 100 101 evaluation tan δ (index) Lambourn abrasion 112 106 111 108105 106 100 102 (index) Tensile strength 25.8 25.5 25.2 25.5 25.2 25.325.6 24.8 at break (MPa)

As is evident from Table 8, the rubber compositions of Examplesincluding the isoprene oligomers in which at least one atom or groupcontained in the trans structural moiety was replaced by another atom orgroup were excellent in low-heat-build-up properties, abrasionresistance, and elongation at break.

As is evident from Table 9, the rubber compositions of Examplesincluding the polyisoprenes in which at least one atom or groupcontained in the trans structural moiety was replaced by another atom orgroup were excellent in low-heat-build-up properties, abrasionresistance, and tensile strength at break.

Next, the following experiment was conducted in order to demonstrate thefact that by maintaining the structure of moiety I in the formula (I) inthe naturally occurring initiating substrate farnesyl diphosphate or thelike, even when a desired structure is introduced in a moiety other thanmoiety I, it is possible to produce isoprene oligomers in the presenceof the naturally occurring oligomer-producing enzyme prenyltransferaseor any enzyme obtained by partial mutation thereof, whereas thisenzymatic reaction does not proceed in the case of using initiatingsubstrates not maintaining the structure of moiety I in the formula (I).

First, the following initiating substrates 1 to 4 not maintaining thestructure of moiety I in the formula (I) were synthesized.

Synthesis of initiating substrate 1 (2E-butenyl diphosphate)

Synthesis was carried out with crotyl alcohol as a starting material.The primary hydroxy group was replaced by chlorine usingN-chlorosuccinimide and dimethyl sulfide in an anhydrous dichloromethanesolvent to obtain a chloride (compound represented by (i) below) (yield:78%). Next, the chloride was diphosphorylated usingtris-tetra-n-butylammonium hydrogen pyrophosphate in anhydrousacetonitrile to obtain a compound represented by (ii) below (initiatingsubstrate 1) as the substance of interest (yield: 50%). The intermediatein each synthesis step and the final product were confirmed by TLC andinstrumental analysis (IR, NMR).

Synthesis of initiating substrate 2 (7-methyl-octa-2E,6E-dienyldiphosphate)

Synthesis was carried out with 3-methyl-2-buten-1-ol as a startingmaterial. The hydroxy group of 3-methyl-2-buten-1-ol was chlorinatedusing N-chlorosuccinimide (NCS) and dimethyl sulfide (DMS) in anhydrousdichloromethane in a nitrogen atmosphere to obtain a chloride (compoundrepresented by (i) below) (yield: 90%). Next, the chloride was reactedwith ethyl cyanoacetate in the presence of lithium hydroxide inanhydrous N,N-dimethylformamide to obtain an ester form (compoundrepresented by (ii) below) (yield: 44%). Next, the ester was hydrolyzedwith potassium hydroxide and methanol to obtain a carboxylic acid(compound represented by (iii) below) (yield: 92%). Next, the carboxylicacid was decarboxylated with dimethyl sulfoxide and common salt toobtain a nitrile form (compound represented by (iv) below) (yield: 40%).The nitrile was reduced with lithium diisobutyl hydride and saturatedammonium chloride in anhydrous dichloromethane to obtain an aldehydeform (compound represented by (v) below) (yield: 89%). The aldehyde wastreated with sodium hydride and ethyl diethylphosphonoacetate inanhydrous tetrahydrofuran to obtain a trans ester form (compoundrepresented by (vi) below) (yield: 84%). Next, the ester was reducedusing lithium diisobutyl hydride and methanol in anhydrousdichloromethane and anhydrous hexane to obtain an alcohol form (compoundrepresented by (vii) below) (yield: 19%). Next, the primary hydroxygroup was replaced by chlorine using N-chlorosuccinimide and dimethylsulfide in an anhydrous dichloromethane solvent at −40° C. or lower toobtain a chloride (compound represented by (viii) below) (yield: 82%).Next, the chloride was diphosphorylated using tris-tetra-n-butylammoniumhydrogen pyrophosphate in anhydrous acetonitrile to obtain a compoundrepresented by (ix) below (initiating substrate 2) as the substance ofinterest (yield: 50%). The intermediate in each synthesis step and thefinal product were confirmed by TLC and instrumental analysis (IR, NMR).

Synthesis of initiating substrate 3(7,11-dimethyl-dodeca-2E,6E,10E-trienyl diphosphate)

Synthesis was carried out with geraniol as a starting material. Thehydroxy group of geraniol was chlorinated using N-chlorosuccinimide(NCS) and dimethyl sulfide (DMS) in anhydrous dichloromethane in anitrogen atmosphere to obtain a chloride (compound represented by (i)below) (yield: 94%). Next, the chloride was reacted with ethylcyanoacetate in the presence of lithium hydroxide in anhydrousN,N-dimethylformamide to obtain an ester form (compound represented by(ii) below) (yield: 34%). Next, the ester was hydrolyzed with potassiumhydroxide and methanol to obtain a carboxylic acid (compound representedby (iii) below) (yield: 95%). Next, the carboxylic acid wasdecarboxylated with dimethyl sulfoxide and common salt to obtain anitrile form (compound represented by (iv) below) (yield: 40%). Thenitrile was reduced with lithium diisobutyl hydride and saturatedammonium chloride in anhydrous dichloromethane to obtain an aldehydeform (compound represented by (v) below) (yield: 91%). The aldehyde wastreated with sodium hydride and ethyl diethylphosphonoacetate inanhydrous tetrahydrofuran to obtain a trans ester form (compoundrepresented by (vi) below) (yield: 82%). Next, the ester was reducedusing lithium diisobutyl hydride and methanol in anhydrousdichloromethane and anhydrous hexane to obtain an alcohol form (compoundrepresented by (vii) below) (yield: 19%). Next, the primary hydroxygroup was replaced by chlorine using N-chlorosuccinimide and dimethylsulfide in an anhydrous dichloromethane solvent to obtain a chloride(compound represented by (viii) below) (yield: 82%). Next, the chloridewas diphosphorylated using tris-tetra-n-butylammonium hydrogenpyrophosphate in anhydrous acetonitrile to obtain a compound representedby (ix) below (initiating substrate 3) as the substance of interest(yield: 42%). The intermediate in each synthesis step and the finalproduct were confirmed by TLC and instrumental analysis (IR, NMR).

Synthesis of initiating substrate 4(7,11,15-trimethyl-hexadeca-2E,6E,10E,14E-tetraenyl diphosphate)

Synthesis was carried out with farnesol as a starting material. Thehydroxy group of farnesol was chlorinated using N-chlorosuccinimide(NCS) and dimethyl sulfide (DMS) in anhydrous dichloromethane in anitrogen atmosphere to obtain a chloride (compound represented by (i)below) (yield: 91%). Next, the chloride was reacted with ethylcyanoacetate in the presence of lithium hydroxide in anhydrousN,N-dimethylformamide to obtain an ester form (compound represented by(ii) below) (yield: 34%). Next, the ester was hydrolyzed with potassiumhydroxide and methanol to obtain a carboxylic acid (compound representedby (iii) below) (yield: 88%). Next, the carboxylic acid wasdecarboxylated with dimethyl sulfoxide and common salt to obtain anitrile form (compound represented by (iv) below) (yield: 40%). Thenitrile was reduced with lithium diisobutyl hydride and saturatedammonium chloride in anhydrous dichloromethane to obtain an aldehydeform (compound represented by (v) below) (yield: 81%). The aldehyde wastreated with sodium hydride and ethyl diethylphosphonoacetate inanhydrous tetrahydrofuran to obtain a trans ester form (compoundrepresented by (vi) below) (yield: 84%). Next, the ester was reducedusing lithium diisobutyl hydride and methanol in anhydrousdichloromethane and anhydrous hexane to obtain an alcohol form (compoundrepresented by (vii) below) (yield: 30%). Next, the primary hydroxygroup was replaced by chlorine using N-chlorosuccinimide and dimethylsulfide in an anhydrous dichloromethane solvent to obtain a chloride(compound represented by (viii) below) (yield: 86%). Next, the chloridewas diphosphorylated using tris-tetra-n-butylammonium hydrogenpyrophosphate in anhydrous acetonitrile to obtain a compound representedby (ix) below (initiating substrate 4) as the substance of interest(yield: 41%). The intermediate in each synthesis step and the finalproduct were confirmed by TLC and instrumental analysis (IR, NMR).

Next, the compound represented by the formula (G), the compoundrepresented by the formula (I), and the compound represented by theformula (Q) were synthesized in the same way as in Production Examples 1to 11.

Next, reaction was performed under conditions shown below usingMicrococcus luteus B-P 26-derived undecaprenyl diphosphate synthase asan enzyme and each of the initiating substrates 1 to 4, the compoundrepresented by the formula (B), the compound represented by the formula(C), the compound represented by the formula (G), the compoundrepresented by the formula (K), and farnesyl diphosphate (FPP). Theresults were indicated in Table 10 by the relative activity of theenzyme on the initiating substrates 1 to 4, the compound represented bythe formula (B), the compound represented by the formula (C), thecompound represented by the formula (G), and the compound represented bythe formula (K) with the activity of the enzyme on farnesyl diphosphateas 100.

A reaction solution containing 500 ng of the enzyme, 50 mM Tris-HClbuffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100, 25 mM2-mercaptoethanol, 12.5 μM of an initiating substrate, and 50 μM [1-¹⁴C]isopentenyl diphosphate was prepared and reacted for 1 hour in a waterbath at 37° C. After the reaction, liquid scintillation counting and TLCquantification were performed to measure the activity of the enzyme oneach initiating substrate.

TABLE 10 Initiating substrate Relative activity (%) Farnesyl diphosphate100 Initiating substrate 1 0.3 Initiating substrate 2 0.6 Initiatingsubstrate 3 1.2 Initiating substrate 4 5.9 Compound of formula (B) 68.4Compound of formula (C) 74.8 Compound of formula (G) 72.9 Compound offormula (K) 40.6

As is evident from the results of Table 10, the enzymatic reactionhardly proceeded in the case of using the initiating substrates 1 to 4not maintaining the structure of moiety I in the formula (I). Incontrast, the enzymatic reaction proceeded in the case of using theinitiating substrates maintaining the structure of moiety I in theformula (I).

Next, the following experiment was conducted using Bacillusstearothermophilus-derived farnesyl diphosphate synthase and Sulfolobusacidocaldarius-derived geranylgeranyl diphosphate synthase in order todemonstrate the fact that even enzymes having prenyltransferaseactivity, derived from organisms other than Micrococcus luteus B-P 26exhibit the same tendencies as described for Micrococcus luteus B-P26-derived undecaprenyl diphosphate synthase, depending on whether ornot the initiating substrate used has maintained the structure of moietyI in the formula (I).

First, Bacillus stearothermophilus-derived farnesyl diphosphate synthasewas prepared.

E. coli BL21 (DE3) was transformed in the same way as above, with aplasmid pET22b containing the base sequence of Bacillusstearothermophilus-derived farnesyl diphosphate synthase (this plasmidis referred to as pET22b/BsFPS). This pET22b/BsFPS was kindly providedby Professor Koyama (Institute of Multidisciplinary Research forAdvanced Materials Tohoku University).

The E. coli BL21(DE3)/pET22b/BsFPS was inoculated into a test tubecontaining 3 mL of an LB medium containing 50 μg/mL ampicillin andshake-cultured at 37° C. for 5 hours. A 1 mL aliquot of the obtainedculture solution was inoculated into a 500-mL Erlenmeyer flaskcontaining 100 mL of an LB medium containing 50 μg/mL ampicillin andshake-cultured at 37° C. for 3 hours. Then, IPTG was added thereto at aconcentration of 0.1 mmol/L, and the bacterial cells were shake-culturedat 30° C. for 18 hours. The culture solution was centrifuged to obtainwet bacterial cells. The wet bacterial cells thus obtained weredisrupted by sonication and then centrifuged. A protein havingprenyltransferase activity (Bacillus stearothermophilus-derived farnesyldiphosphate synthase) was purified from the obtained supernatant usingHisTrap (manufactured by Amersham Biosciences Corp.). The purificationof the purified protein was confirmed by SDS-PAGE.

Next, Sulfolobus acidocaldarius-derived geranylgeranyl diphosphatesynthase was prepared.

E. coli BL21 (DE3) was transformed in the same way as above, with aplasmid pET22b containing the base sequence of Sulfolobusacidocaldarius-derived geranylgeranyl diphosphate synthase (this plasmidis referred to as pET22b/SaGGPS). This pET22b/SaGGPS was kindly providedby Professor Tokuzo Nishino (School of Engineering, Tohoku University).

The E. coli BL21(DE3)/pET22b/SaGGPS was inoculated into a test tubecontaining 3 mL of an LB medium containing 50 μg/mL ampicillin andshake-cultured at 37° C. for 5 hours. A 1 mL aliquot of the obtainedculture solution was inoculated into a 500-mL Erlenmeyer flaskcontaining 100 mL of an LB medium containing 50 μg/mL ampicillin andshake-cultured at 37° C. for 3 hours. Then, IPTG was added thereto at aconcentration of 0.1 mmol/L, and the bacterial cells were shake-culturedat 30° C. for 18 hours. The culture solution was centrifuged to obtainwet bacterial cells. The wet bacterial cells thus obtained weredisrupted by sonication and then centrifuged. A protein havingprenyltransferase activity (Sulfolobus acidocaldarius-derivedgeranylgeranyl diphosphate synthase) was purified from the obtainedsupernatant using HisTrap (manufactured by Amersham Biosciences Corp.).The purification of the purified protein was confirmed by SDS-PAGE.

Reaction was performed under conditions shown below using the obtainedBacillus stearothermophilus-derived farnesyl diphosphate synthase andeach of the initiating substrates 1 to 4, the compound represented bythe formula (F), the compound represented by the formula (I), thecompound represented by the formula (Q), and geranyl diphosphate (GPP)whose structure is shown below. The results were indicated in Table 11by the relative activity of the enzyme on the initiating substrates 1 to4, the compound represented by the formula (F), the compound representedby the formula (I), and the compound represented by the formula (Q) withthe activity of the enzyme on geranyl diphosphate as 100.

A reaction solution containing 500 ng of the purified enzyme, 50 mMTris-HCl buffer (pH 8.5), 40 mM magnesium chloride, 50 mM ammoniumchloride, 40 mM Triton X-100, 25 mM 2-mercaptoethanol, 12.5 μM of aninitiating substrate, and 50 μM [1-¹⁴C] isopentenyl diphosphate wasprepared and reacted for 1 hour in a water bath at 55° C. After thereaction, liquid scintillation counting and TLC quantification wereperformed to measure the activity of the enzyme on each initiatingsubstrate.

TABLE 11 Initiating substrate Relative activity (%) Geranyl diphosphate100 Initiating substrate 1 1.4 Initiating substrate 2 3.2 Initiatingsubstrate 3 2.1 Initiating substrate 4 0.7 Compound of formula (F) 52.3Compound of formula (I) 44.1 Compound of formula (Q) 54.3

Likewise, reaction was performed under conditions shown below usingSulfolobus acidocaldarius-derived geranylgeranyl diphosphate synthaseand each of the initiating substrates 1 to 4, the compound representedby the formula (B), the compound represented by the formula (C), thecompound represented by the formula (G), the compound represented by theformula (K), the compound represented by the formula (F), the compoundrepresented by the formula (I), the compound represented by the formula(Q), farnesyl diphosphate (FPP), and geranyl diphosphate (GPP). Theresults were indicated in Tables 12 and 13 by the relative activity ofthe enzyme on the initiating substrates 1 to 4, the compound representedby the formula (B), the compound represented by the formula (C), thecompound represented by the formula (G), the compound represented by theformula (K), the compound represented by the formula (F), the compoundrepresented by the formula (I), and the compound represented by theformula (Q) with the activity of the enzyme on farnesyl diphosphate orgeranyl diphosphate as 100.

A reaction solution containing 500 ng of the purified enzyme, 50 mMTris-HCl buffer (pH 5.8), 40 mM magnesium chloride, 50 mM ammoniumchloride, 40 mM Triton X-100, 25 mM 2-mercaptoethanol, 12.5 μM of aninitiating substrate, and 50 μM [1-¹⁴C] isopentenyl diphosphate wasprepared and reacted for 1 hour in a water bath at 55° C. After thereaction, liquid scintillation counting and TLC quantification wereperformed to measure the activity of the enzyme on each initiatingsubstrate.

TABLE 12 Initiating substrate Relative activity (%) Geranyl diphosphate100 Initiating substrate 1 0.4 Initiating substrate 2 0.4 Initiatingsubstrate 3 0.6 Initiating substrate 4 0.8 Compound of formula (F) 52.3Compound of formula (I) 44.1 Compound of formula (Q) 54.3

TABLE 13 Initiating substrate Relative activity (%) Farnesyl diphosphate100 Initiating substrate 1 2.3 Initiating substrate 2 2.5 Initiatingsubstrate 3 3.9 Initiating substrate 4 1.4 Compound of formula (B) 70.6Compound of formula (C) 37.3 Compound of formula (G) 70.4 Compound offormula (K) 38.6

As is evident from the results of Tables 11 to 13, the enzymaticreactions of Bacillus stearothermophilus-derived farnesyl diphosphatesynthase and Sulfolobus acidocaldarius-derived geranylgeranyldiphosphate synthase hardly proceeded in the case of using theinitiating substrates 1 to 4 not maintaining the structure of moiety Iin the formula (I), as in use of Micrococcus luteus B-P 26-derivedundecaprenyl diphosphate synthase. In contrast, the enzymatic reactionsproceeded in the case of using the initiating substrates maintaining thestructure of moiety I in the formula (I).

The results of Tables 1 to 3 and 10 to 13 have demonstrated that bymaintaining the structure of moiety I in the formula (I) in thenaturally occurring initiating substrate farnesyl diphosphate, geranyldiphosphate, or the like, even when a desired structure is introduced ina moiety other than moiety I, it is possible to produce isopreneoligomers in the presence of an enzyme having prenyltransferaseactivity, which is a naturally occurring oligomer-producing enzyme, orany enzyme obtained by partial mutation thereof.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: Base sequence of Micrococcus luteus B-P 26-derivedundecaprenyl diphosphate synthase (wild-type enzyme)

SEQ ID NO: 2: Amino acid sequence of Micrococcus luteus B-P 26-derivedundecaprenyl diphosphate synthase (wild-type enzyme)

SEQ ID NO: 3: Base sequence of variant enzyme N31A

SEQ ID NO: 4: Amino acid sequence of variant enzyme N31A

SEQ ID NO: 5: Base sequence of variant enzyme N77A

SEQ ID NO: 6: Amino acid sequence of variant enzyme N77A

SEQ ID NO: 7: Base sequence of variant enzyme L91N

SEQ ID NO: 8: Amino acid sequence of variant enzyme L91N

SEQ ID NO: 9: Base sequence of variant enzyme L91D

SEQ ID NO: 10: Amino acid sequence of variant enzyme L91D

SEQ ID NO: 11: Base sequence of variant enzyme N31Q

SEQ ID NO: 12: Amino acid sequence of variant enzyme N31Q

SEQ ID NO: 13: Base sequence of variant enzyme N77Q

SEQ ID NO: 14: Amino acid sequence of variant enzyme N77Q

SEQ ID NO: 15: Base sequence of variant enzyme L91G

SEQ ID NO: 16: Amino acid sequence of variant enzyme L91G

SEQ ID NO: 17: Base sequence of variant enzyme L91K

SEQ ID NO: 18: Amino acid sequence of variant enzyme L91K

SEQ ID NO: 19: Base sequence of variant enzyme F95A

SEQ ID NO: 20: Amino acid sequence of variant enzyme F95A

SEQ ID NO: 21: Base sequence of variant enzyme F95W

SEQ ID NO: 22: Amino acid sequence of variant enzyme F95W

SEQ ID NO: 23: Sense primer for preparation of variant enzyme N31A

SEQ ID NO: 24: Antisense primer for preparation of variant enzyme N31A

SEQ ID NO: 25: Sense primer for preparation of variant enzyme N77A

SEQ ID NO: 26: Antisense primer for preparation of variant enzyme N77A

SEQ ID NO: 27: Sense primer for preparation of variant enzyme L91N

SEQ ID NO: 28: Antisense primer for preparation of variant enzyme L91N

SEQ ID NO: 29: Sense primer for preparation of variant enzyme L91D

SEQ ID NO: 30: Antisense primer for preparation of variant enzyme L91D

SEQ ID NO: 31: Sense primer for preparation of variant enzyme N31Q

SEQ ID NO: 32: Antisense primer for preparation of variant enzyme N31Q

SEQ ID NO: 33: Sense primer for preparation of variant enzyme N77Q

SEQ ID NO: 34: Antisense primer for preparation of variant enzyme N77Q

SEQ ID NO: 35: Sense primer for preparation of variant enzyme L91G

SEQ ID NO: 36: Antisense primer for preparation of variant enzyme L91G

SEQ ID NO: 37: Sense primer for preparation of variant enzyme L91K

SEQ ID NO: 38: Antisense primer for preparation of variant enzyme L91K

SEQ ID NO: 39: Sense primer for preparation of variant enzyme F95A

SEQ ID NO: 40: Antisense primer for preparation of variant enzyme F95A

SEQ ID NO: 41: Sense primer for preparation of variant enzyme F95W

SEQ ID NO: 42: Antisense primer for preparation of variant enzyme F95W

The invention claimed is:
 1. An isoprene oligomer comprising asubstituted trans structural moiety and a cis structural moiety, whereinat least one atom or group of an unsubstituted trans structural moiety Aof a preliminary isoprene oligomer having the following formula (1)selected from a hydrogen atom, a methyl group, a methylene group, acarbon atom, or a methine group is replaced by a different atom or groupto provide the substituted trans structural moiety, and the differentatom or group is selected from the group consisting of a nitrogen atom,an oxygen atom, a sulfur atom, a silicon atom, a carbon atom, an acetoxygroup, an alkoxy group, a hydroxy group, an aryl group, an alkyl group,an acetyl group, an N-alkyl-acetamino group, and an azide group:

wherein n represents an integer from 1 to 10; m represents an integerfrom 1 to 30; the preliminary isoprene oligomer has a cis structuralmoiety B; and Y represents a hydroxy group, a formyl group, a carboxygroup, an ester group, a carbonyl group, or a group having the followingformula (2):


2. The isoprene oligomer according to claim 1, further comprising asubstituted trans group-containing moiety II, an unsubstituted transmoiety III, and the remainder of the isoprene oligomer being a cisgroup-containing moiety, wherein at least one atom or group of anunsubstituted trans group-containing moiety II in the following formula(1-1) selected from a hydrogen atom, a methyl group, a methylene group,a carbon atom, or a methine group is replaced by a different atom orgroup to provide the substituted trans group-containing moiety II; thedifferent atom or group is selected from the group consisting of anitrogen atom, an oxygen atom, a sulfur atom, a silicon atom, a carbonatom, an acetoxy group, an alkoxy group, a hydroxy group, an aryl group,an alkyl group, an acetyl group, an N-alkyl-acetamino group, and anazide group,


3. The isoprene oligomer according to claim 1 or 2, wherein the transstructural moiety A has any of the following formulas (a) to (s):


4. The isoprene oligomer according to claim 1, wherein the isopreneoligomer is biosynthesized using an allylic diphosphate and isopentenyldiphosphate; the allylic diphosphate has the following formula (3),wherein at least 1 atom or group in the isoprene units in formula (3)selected from a hydrogen atom, a methyl group, a methylene group, acarbon atom, or a methine group is replaced by a different atom orgroup, and the different atom or group is selected from the groupconsisting of a nitrogen atom, an oxygen atom, a sulfur atom, a siliconatom, a carbon atom, an acetoxy group, an alkoxy group, a hydroxy group,an aryl group, an alkyl group, an acetyl group, an N-alkyl-acetaminogroup, and an azide group:

wherein p represents an integer from 1 to
 10. 5. The isoprene oligomeraccording to claim 4, wherein the biosynthesis is carried out using anenzyme that shows prenyltransferase activity.
 6. The isoprene oligomeraccording to claim 5, wherein the enzyme that shows prenyltransferaseactivity is any of the following proteins: [1] a protein comprising anamino acid sequence having any of the following SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, and 22; [2] a protein comprising an amino acidsequence that is derived from the amino acid sequence having any of thefollowing SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 bysubstitution, deletion, insertion, or addition of 1 or more amino acids,and possessing the ability to catalyze a reaction between an allylicdiphosphate and isopentenyl diphosphate, wherein the allylic diphosphatehas the following formula (3), wherein at least 1 atom or group in theisoprene units in formula (3) is replaced by another atom or group; or[3] a protein comprising an amino acid sequence that shows 45% or highersequence identity to the amino acid sequence having any of the followingSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 and possessingthe ability to catalyze a reaction between an allylic diphosphate andisopentenyl diphosphate, wherein the allylic diphosphate has thefollowing formula (3), wherein at least 1 atom or group in the isopreneunits in formula (3) is replaced by another atom or group:

wherein p represents an integer from 1 to
 10. 7. A process for producingthe isoprene oligomer according to claim 1, comprising biosynthesis ofthe isoprene oligomer in presence of allylic diphosphate and isopentenyldiphosphate, wherein the allylic diphosphate has the following formula(3), wherein at least 1 atom or group in the isoprene units in formula(3) is replaced by another atom or group:

wherein p represents an integer from 1 to
 10. 8. The process forproducing the isoprene oligomer according to claim 7, wherein thebiosynthesis is carried out in presence of an enzyme showingprenyltransferase activity.